WO2023078435A1 - 叶酸在预防、诊断和治疗遗传性、感染性或过敏性疾病中的应用 - Google Patents

叶酸在预防、诊断和治疗遗传性、感染性或过敏性疾病中的应用 Download PDF

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WO2023078435A1
WO2023078435A1 PCT/CN2022/130161 CN2022130161W WO2023078435A1 WO 2023078435 A1 WO2023078435 A1 WO 2023078435A1 CN 2022130161 W CN2022130161 W CN 2022130161W WO 2023078435 A1 WO2023078435 A1 WO 2023078435A1
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folic acid
biliary atresia
vitamin
caused
neonatal
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PCT/CN2022/130161
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English (en)
French (fr)
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张玉霞
温哲
徐艳慧
陈章华
方榕丽
陈西茜
宁禹
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广州市妇女儿童医疗中心
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Definitions

  • the invention belongs to the technical field of biomedicine, and specifically relates to the application of folic acid in the prevention, diagnosis and treatment of hereditary, infectious or allergic diseases.
  • Biliary atresia (hereinafter sometimes referred to as "BA") is a common disease that causes obstructive jaundice in infants and young children.
  • BA Biliary atresia
  • viruses known to be associated with the occurrence of BA include human herpesvirus, cytomegalovirus, rotavirus, reovirus, etc.
  • autoimmune reactions Stimulate autoimmune reactions that cause pathological changes such as apoptosis or necrosis of bile duct epithelial cells, bile duct damage, inflammation, and fibrosis.
  • the prognosis is poor and the mortality rate is high.
  • biliary atresia The basic pathological changes of biliary atresia are, for example, progressive inflammation of intrahepatic and extrahepatic bile ducts and liver fibrosis.
  • the development of liver fibrosis is faster and more aggressive than other adult diseases, although extrahepatic biliary obstruction can be partially relieved by Kasai operation Symptoms can delay the progression of the disease, but most children still develop progressively due to postoperative intrahepatic bile duct inflammation, eventually leading to cirrhosis, portal hypertension, and even liver failure, becoming a life-threatening serious disease in children.
  • Biliary atresia is currently the leading cause of liver transplantation in childhood.
  • biliary atresia disease is indistinguishable from other infants and young children with persistent jaundice. It needs to be diagnosed clinically through invasive methods such as liver puncture. In addition to Kasai surgery, liver transplantation and other surgical methods, there are no effective drugs. Treatment, in our country, more than 70% of children often die due to the long time of diagnosis and the delay of the optimal treatment window of surgery.
  • Neonatal infectious disease also known as neonatal infection
  • neonatal infection is a common neonatal disease and an important factor in neonatal death, including lung infection, umbilical cord infection, brain infection, skin infection, urinary Systemic infection, oral infection, etc.
  • infection routes include maternal intrauterine infection, intrapartum infection, postnatal infection, etc.
  • Folic acid also known as vitamin M, vitamin Bc, and vitamin B9, is a water-soluble vitamin.
  • the demand for folic acid in normal people is 200-400 ⁇ g/d.
  • the World Health Organization recommends at least 200 ⁇ g/d for adults, and 400 ⁇ g for pregnant women and breast milk. /d, generally 20 times more than the minimum requirement of adults will not cause poisoning.
  • folic acid plays an important physiological role in the body, including maintaining the production and function of red blood cells, but there is no report on the effect of folic acid on biliary atresia.
  • the present invention has a comprehensive understanding of the pathogenesis of genetic, infectious or allergic diseases, intestinal dysfunction associated with diseases, complications such as liver fibrosis, and related diseases such as biliary atresia, cholangitis, jaundice, viral infections, etc.
  • the diagnosis, prevention and treatment of the disease have been deeply researched, and the following results have been obtained:
  • the present invention finds that folic acid is significantly reduced in the serum of BA patients and can be used as a marker for the diagnosis or auxiliary diagnosis of BA.
  • folic acid in the virus-infected biliary atresia mouse model can significantly increase the body weight of the mice, reduce the jaundice rate of the mice, improve the liver function of the mice, and inhibit the production of inflammatory cytokines S100a8, S100a9 and IFN- ⁇ in the intestine or liver It promotes the expression of myeloid-derived suppressor cell characteristic transcription factor Nox2; at the same time, folic acid can also significantly improve intestinal inflammation, regulate iron ion metabolism, correct intestinal flora disorder, and reduce liver and intestinal tissue damage.
  • folic acid can treat biliary atresia, cholangitis, liver fibrosis, gastrointestinal flora disorder, and inflammatory bowel disease by improving anti-inflammation, regulating iron ion metabolism, correcting intestinal flora disorder, and improving liver damage. , Diagnosis and prevention of viral infection. Folic acid can also reduce the total bilirubin concentration and direct bilirubin concentration in patients with biliary atresia, so as to prevent and/or treat cholangitis.
  • folic acid supplementation can also correct systemic iron overload and luminal iron deficiency, inhibit eosinophil infiltration in the small intestine, inhibit the production of IgG-Ro/SSA autoantibodies, increase the ratio of IgM/IgG4, and at the same time, prolong the period of autoimmunity and improve Liver function;
  • supplementing calcium folinate (CF) can reduce the content of INF ⁇ and ROS in the liver, reduce the incidence of jaundice and improve neonatal growth; increase the expression of Dhfr, Mthfr, Nrf2, Slc40a1 in the small intestine , so as to restore folic acid metabolism, oxidative stress and iron homeostasis; reduce the content of IFN- ⁇ in CD4 + T cells and CD8 + T cells in the liver, and the content of IgG1 in plasma cells; reduce the infiltration of lymphocytes around the portal area of the liver; Increase the structural integrity of the hepatic
  • the present invention discloses for the first time that at least one of folic acid, S100a8, S100a9, IFN- ⁇ , Nox2 can be used as a diagnosis of biliary atresia Or the use of markers for auxiliary diagnosis.
  • the present invention also discloses that folic acid or its derivatives alone combined with other vitamin B are used for the prevention and treatment of biliary atresia, cholangitis, jaundice, food allergy, bacterial infection, virus infection, necrotizing enterocolitis, and for the prevention and treatment of biliary atresia
  • folic acid or its derivatives alone combined with other vitamin B are used for the prevention and treatment of biliary atresia, cholangitis, jaundice, food allergy, bacterial infection, virus infection, necrotizing enterocolitis, and for the prevention and treatment of biliary atresia
  • the resulting jaundice, liver inflammatory disease, liver function damage, and intestinal repair all showed significant effects.
  • the purpose of the first aspect of the present invention is to provide the application of folic acid or its derivatives in the preparation of medicines.
  • the object of the second aspect of the present invention is to provide the use of folic acid or its derivatives in the preparation of in vitro non-therapeutic products.
  • the purpose of the third aspect of the present invention is to provide markers for diagnosis or auxiliary diagnosis of biliary atresia disease.
  • the purpose of the fourth aspect of the present invention is to provide the application of the reagent for detecting the marker of the third aspect of the present invention in the preparation of products for diagnosis or auxiliary diagnosis of biliary atresia.
  • the purpose of the fifth aspect of the present invention is to provide a kit for diagnosis or auxiliary diagnosis of biliary atresia disease.
  • the purpose of the sixth aspect of the present invention is to provide the use of folic acid or its derivatives in the preparation of medicaments for preventing and/or treating viral infections.
  • the purpose of the sixth aspect of the present invention is to provide the use of folic acid or its derivatives in the preparation of medicaments for preventing and/or treating neonatal infection.
  • the purpose of the seventh aspect of the present invention is to provide the use of folic acid or its derivatives in the preparation of drugs for the prevention and/or treatment of diseases caused by enterohepatic circulation disorders.
  • the object of the eighth aspect of the present invention is to provide the use of folic acid or its derivatives in the preparation of medicaments for preventing and/or treating inflammatory bowel disease.
  • the purpose of the ninth aspect of the present invention is to provide the application of folic acid or its derivatives in the preparation of products for regulating intestinal flora.
  • the purpose of the tenth aspect of the present invention is to provide the use of folic acid or its derivatives in the preparation of medicaments for the prevention and/or treatment of diseases caused by increased expression of S100a8 or S100a9.
  • the object of the eleventh aspect of the present invention is to provide the use of folic acid or its derivatives in the preparation of products for preventing and/or treating iron deficiency anemia.
  • the object of the twelfth aspect of the present invention is to provide the use of folic acid or its derivatives in the preparation of medicines for preventing and/or treating diseases caused by increased expression of IFN- ⁇ .
  • the object of the thirteenth aspect of the present invention is to provide a preparation method of a product containing folic acid or its derivatives.
  • the object of the fourteenth aspect of the present invention is to provide a medicine.
  • the object of the fifteenth aspect of the present invention is to provide a diagnostic method.
  • the object of the sixteenth aspect of the present invention is to provide a method for preventing and/or treating diseases.
  • the first aspect of the present invention provides the application of folic acid or its derivatives in the preparation of medicine
  • the drug has at least one function in (a1) to (a9):
  • the second aspect of the present invention provides the application of folic acid or its derivatives in any one of (b1)-(b5);
  • the inflammatory factors comprise at least one of S100a8, S100a9 and IFN- ⁇ .
  • the myeloid-derived suppressor cell characteristic transcription factor comprises Nox2.
  • the folate transporter comprises SLC46A1.
  • the third aspect of the present invention provides a marker for diagnosis or auxiliary diagnosis of biliary atresia disease, comprising at least one of folic acid, S100a8, S100a9, Nox2, and IFN- ⁇ .
  • the markers include:
  • said S100a8, S100a9, Nox2 and IFN- ⁇ include protein and/or mRNA.
  • the marker is from body fluid, blood, tissue, cell or excrement; further preferably, the marker is from blood, tissue or excrement.
  • said tissue comprises liver, intestinal tract, embryonic liver, embryonic intestine.
  • the excrement includes feces and urine.
  • the blood is whole blood, serum or plasma; further is serum.
  • said folic acid is from blood.
  • the S100a8, S100a9, Nox2 and IFN- ⁇ are from tissues; further preferably, the S100a8, S100a9, Nox2 and IFN- ⁇ are from liver tissue or intestinal tissue; still more preferably, the S100a8, S100a9 , Nox2, and IFN- ⁇ come from intestinal tissue.
  • test subject when the test subject satisfies at least one of (c1)-(c5), it is diagnosed as a patient with biliary atresia disease;
  • the reference level is the level of subjects of the same age who do not suffer from biliary atresia; the subjects of the same age are healthy people or patients with choledochal cyst.
  • the fourth aspect of the present invention provides the application of the reagent for detecting the marker of the third aspect of the present invention in the preparation of products for diagnosis or auxiliary diagnosis of biliary atresia.
  • the products include reagents, kits, gene chips, and protein chips.
  • the product is used to perform at least one of the following detection methods:
  • the product contains primers and/or detection probes for detecting the marker and/or internal reference mRNA.
  • the product further comprises: sample processing reagents, including but not limited to sample lysing reagents, sample purification reagents and/or sample nucleic acid extraction reagent.
  • the product further comprises: RNA extraction reagent, dNTP, DNA polymerase, double-strand specific fluorescent dye and one of the following in water or more.
  • the product when the detection marker is S100a8mRNA, the product includes primers for detecting S100a8mRNA, and the nucleotide sequences of the primers are shown in SEQ ID NO.1 and SEQ ID NO.2.
  • the product when the detection marker is S100a9mRNA, the product includes primers for detecting S100a9mRNA, and the nucleotide sequences of the primers are shown in SEQ ID NO.3 and SEQ ID NO.4.
  • the product when the detection marker is IFN- ⁇ mRNA, the product includes primers for detecting IFN- ⁇ mRNA, and the nucleotide sequences of the primers are shown in SEQ ID NO.5 and SEQ ID NO.6.
  • the product when the detection marker is Nox2 mRNA, the product includes primers for detecting Nox2 mRNA, and the nucleotide sequences of the primers are shown in SEQ ID NO.7 and SEQ ID NO.8.
  • test sample or test sample of the product is from body fluid, blood, tissue, cell or excrement of the subject to be tested; further preferably, the marker is from blood, tissue or excrement.
  • said tissue comprises liver, intestinal tract, embryonic liver, embryonic intestine.
  • the excrement includes feces and urine.
  • the blood is whole blood, serum or plasma, dried blood spots; further serum, dried blood spots.
  • the subject to be tested is an adult or a child.
  • the children include newborns within 28 days of birth, infants under 1 year old, infants aged 1-6 years old, and children aged 6-8 years old.
  • the adult is selected from a pregnant female adult, a perinatal female adult or a lactating female adult.
  • test subject when the test subject satisfies at least one of (c1)-(c5), it is diagnosed as a patient with biliary atresia disease;
  • the reference level is the level of subjects of the same age who do not suffer from biliary atresia; the subjects of the same age are healthy people or patients with choledochal cyst.
  • the fifth aspect of the present invention is to provide a kit for diagnosing or assisting the diagnosis of biliary atresia disease, comprising a reagent for detecting the marker of the third aspect of the present invention.
  • the product contains primers and/or detection probes for detecting the marker and/or internal reference mRNA.
  • the product further comprises: sample processing reagents, including but not limited to sample lysing reagents, sample purification reagents and/or sample nucleic acid extraction reagent.
  • the product further comprises: RNA extraction reagent, dNTP, DNA polymerase, double-strand specific fluorescent dye and one of the following in water or more.
  • the product when the detection marker is S100a8mRNA, the product includes primers for detecting S100a8mRNA, and the nucleotide sequences of the primers are shown in SEQ ID NO.1 and SEQ ID NO.2.
  • the product when the detection marker is S100a9mRNA, the product includes primers for detecting S100a9mRNA, and the nucleotide sequences of the primers are shown in SEQ ID NO.3 and SEQ ID NO.4.
  • the product when the detection marker is IFN- ⁇ mRNA, the product includes primers for detecting IFN- ⁇ mRNA, and the nucleotide sequences of the primers are shown in SEQ ID NO.5 and SEQ ID NO.6.
  • the product when the detection marker is Nox2 mRNA, the product includes primers for detecting Nox2 mRNA, and the nucleotide sequences of the primers are shown in SEQ ID NO.7 and SEQ ID NO.8.
  • test sample or test sample of the product is from body fluid, blood, tissue, cell or excrement of the subject to be tested; further preferably, the marker is from blood, tissue or excrement.
  • said tissue comprises liver, intestinal tract, embryonic liver, embryonic intestine.
  • the excrement includes feces and urine.
  • the blood is whole blood, serum or plasma, dried blood spots; further serum, dried blood spots.
  • the subject to be tested is an adult or a child.
  • the children include newborns within 28 days of birth, infants under 1 year old, infants aged 1-6 years old, and children aged 6-8 years old.
  • the adult is selected from a pregnant female adult, a perinatal female adult or a lactating female adult.
  • test subject when the test subject satisfies at least one of (c1)-(c5), it is diagnosed as a patient with biliary atresia disease;
  • the reference level is the level of subjects of the same age who do not suffer from biliary atresia; the subjects of the same age are healthy people or patients with choledochal cyst.
  • the sixth aspect of the present invention provides the use of folic acid or its derivatives in the preparation of medicaments for preventing and/or treating viral infections.
  • the seventh aspect of the present invention provides the use of folic acid or its derivatives in the preparation of drugs for the prevention and/or treatment of diseases caused by enterohepatic circulation disorders.
  • the eighth aspect of the present invention provides the use of folic acid or its derivatives in the preparation of medicaments for preventing and/or treating inflammatory bowel disease.
  • the ninth aspect of the present invention provides the use of folic acid or its derivatives in the preparation of products for regulating intestinal flora.
  • said products include food, nutraceuticals and pharmaceuticals.
  • the product is used for the prevention, treatment or auxiliary treatment of Candida albicans enteritis, staphylococcal enteritis, acute necrotic enteritis caused by Clostridium perfringens, (neonatal) necrotizing enterocolitis, septicemia, Pseudomonas aeruginosa intestinal infection, Proteus intestinal infection, Klebsiella pneumoniae intestinal infection and other intestinal inflammatory diseases or infectious diseases; and reduce the risk of premature birth caused by the above diseases.
  • the tenth aspect of the present invention provides the use of folic acid or its derivatives in the preparation of drugs for the prevention and/or treatment of diseases caused by the increased expression of S100a8 and/or S100a9.
  • the eleventh aspect of the present invention provides the use of folic acid or its derivatives in the preparation of products for preventing and/or treating iron deficiency anemia.
  • said products include food, nutraceuticals and pharmaceuticals.
  • the twelfth aspect of the present invention provides the use of folic acid or its derivatives in the preparation of medicines for diseases caused by increased expression of IFN- ⁇ .
  • the thirteenth aspect of the present invention provides a method for preparing a product containing folic acid or its derivatives, which is obtained by mixing folic acid or its derivatives with auxiliary materials in proportion.
  • the preparation method further includes preparing the obtained mixture into corresponding forms or dosage forms according to requirements.
  • said products include food, nutraceuticals and pharmaceuticals.
  • a medicine comprising: folic acid or its derivatives
  • the other active ingredients include at least one of vitamin B6 and vitamin B12.
  • the medicine includes: folic acid or its derivatives; and at least one of vitamin B6, vitamin B12, vitamin A, vitamin D, vitamin E, vitamin K, vitamin C, nicotinamide, zinc oxide, and taurine .
  • the drug comprises folic acid or its derivatives and nicotinamide.
  • the drug comprises folic acid or its derivatives and vitamin E.
  • the medicament comprises folic acid or its derivatives and vitamin A and vitamin E.
  • the drug has at least one function in (a1) to (a9):
  • the fourteenth aspect of the present invention provides a method for diagnosing or assisting in the diagnosis of biliary atresia, comprising at least one of the following steps:
  • a fifteenth aspect of the present invention provides a method, comprising at least one of the following steps:
  • the method is used for at least one of (a1) to (a16):
  • the method includes at least one of the following steps:
  • the method is used for at least one of (a1) to (a9):
  • the molecular formula of folic acid is C 19 H 19 N 7 O 6 , the molecular weight is 441.4, and the CAS is 59-30-3.
  • the derivatives include pharmaceutically acceptable salts, esters, hydrates, solvates, polymorphs, tautomers, prodrugs and functional equivalents of folic acid; further preferably, the derivatives
  • the substances include any one of (a1) to (a2): (a1) folinic acid, dihydrofolate, tetrahydrofolate, 5-methyltetrahydrofolate, 5,10-methylenetetrahydrofolate, 5,10 -Methylenetetrahydrofolate, 5,10-formyltetrahydrofolate, 5-formyltetrahydrofolate, 10-formyltetrahydrofolate, and 10-methyltetrahydrofolate; (a2)(a1) Pharmaceutically acceptable salts, including but not limited to calcium, sodium, zinc, arginine, choline, acetylcholine, N-methylaminoethanol, 2-amino-2-methyl-propanol, 1,1- Dimethylbiguanide, phenethylbig
  • the derivative comprises at least one of a pharmaceutically acceptable salt of folic acid, folinic acid, and a pharmaceutically acceptable salt of folinic acid.
  • the cholangitis comprises bacterial cholangitis and viral cholangitis.
  • the neonatal infection includes: at least one of bacterial infection and viral infection.
  • the virus comprises: at least one of DNA virus and RNA virus.
  • the RNA virus comprises: at least one of antisense RNA virus and double-stranded RNA virus.
  • the neonatal infectious disease comprises: a disease caused by excessive activation of type I interferon pathway.
  • the neonatal infectious disease includes: neonatal hepatitis syndrome, neonatal sepsis, neonatal pneumonia, and gastrointestinal diseases caused by neonatal infection.
  • the bacterial infectious disease includes Escherichia coli, Staphylococcus aureus or hemolytic streptococcus.
  • the viral infectious diseases include cytomegalovirus (CMV) infection, rotavirus infection, and herpes virus infection; wherein, the viral infectious diseases also include viral diarrhea.
  • CMV cytomegalovirus
  • the dosage form of the medicine is a dosage form suitable for children or a dosage form suitable for adults.
  • the children include newborns within 28 days of birth, infants under 1 year old, infants aged 1-6 years old, and children aged 6-18 years old.
  • said adult includes a pregnant female adult, a perinatal female adult and a lactating female adult.
  • the dosage forms include capsules, tablets, microcapsule preparations, injections, suppositories, sprays, powders, soft capsules, dripping pills, honeyed pills, pills, granules, honey paste, sustained and controlled release preparations, Oral liquid formulations, injections, chewable tablets, buccal tablets, transdermal patches, and effervescent tablets.
  • the unit dose of the folic acid or its derivatives in the drug is 0.1 mg-1 g.
  • the unit dose of the folic acid or its derivatives in the drug is 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg or 1.0 mg.
  • the unit dose of the folic acid or its derivatives in the medicament is 0.2 mg, 0.4 mg, 0.8 mg, or 1.0 mg.
  • the medicament further comprises other active ingredients, the other active ingredients being vitamin B, vitamin C, nicotinamide, zinc oxide, vitamin A, vitamin E, homocysteine, glutathione and taurine
  • vitamin B protects the liver
  • vitamin C also known as L-ascorbic acid
  • niacinamide also known as nicotinamide
  • zinc oxide is a kind of zinc oxide
  • vitamin A is a fat-soluble vitamin
  • vitamin E also known as tocopherol or pregnanol
  • homocysteine also known as Homocysteine is a sulfur-containing amino acid
  • glutathione (GSH) is a tripeptide containing a ⁇ -amide bond and a sulfhydryl group, which is composed of glutamic acid, cysteine and glycine, and has antioxidant properties.
  • detoxification; taurine protects
  • the vitamin B is vitamin B1 (sulfammonium), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine), vitamin B7 (biotin ) and at least one of vitamin B12 (cobalamin).
  • the drug includes: folic acid or its derivatives; and at least one of vitamin B6, vitamin B12, vitamin A, vitamin E, vitamin C, nicotinamide, zinc oxide, and taurine .
  • the medicament further comprises pharmaceutical excipients, which include diluents, fillers, excipients, excipients, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption One or more of carrier, lubricant and fragrance.
  • pharmaceutical excipients include diluents, fillers, excipients, excipients, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption One or more of carrier, lubricant and fragrance.
  • the carrier or excipient can be selected from one or more of lactose hydrate, microcrystalline cellulose, mannitol, sodium citrate, calcium phosphate, glycine and starch; One or more of polyvidone, copovidone, sodium starch glycolate, croscarmellose sodium and specific complex silicate; the binder can be selected from polyvinylpyrrolidone, hydroxypropyl One or more of methylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin, and gum arabic.
  • the viruses in the viral infection include but are not limited to human herpesvirus, cytomegalovirus, rotavirus and reovirus.
  • the diseases caused by the enterohepatic circulation disorder include hypercholesterolemia, cholesterol gallstones, jaundice and liver fibrosis.
  • the inflammatory bowel disease includes undifferentiated colitis, ulcerative colitis and Crohn's disease.
  • the diseases caused by the increased expression of S100a8 and/or S100a9 include but are not limited to systemic lupus erythematosus (systemiclupus erythematosus, SLE), rheumatoid arthritis (rheumatoid arthritis, RA), inflammatory bowel disease ((inflammatory bowel disease) , IBD), primary glomerular disease, systemic sclerosis (SSc), multiple sclerosis (MS, multiple sclerosis, experimental autoimmune encephalomyelitis, experimental autoimmune encephalomye-litis EAE), COPD Chronic obstructive pulmonary disease, bone marrow leukopoiesis, myelodysplastic syndrome, tumorigenesis, gastric cancer, breast cancer, melanoma, periodontal inflammatory disease, obesity, type 2 diabetes (type2diabetes mellitus, T2DM) and its complications, heart disease Vascular disease (myocyte dysfunction, myocardial infarction, atherosclerosis) and skin
  • the iron deficiency anemia comprises iron deficiency anemia in children.
  • the diseases caused by the increased expression of IFN- ⁇ include autoimmune diseases in children or adults.
  • the autoimmune diseases include but are not limited to: type I diabetes, aplastic anemia (AA), rheumatoid arthritis (rheumatoid arthritis, RA), systemic lupus erythematosus (systemic lupus erythematosus, SLE), autoimmune Autoimmune thyroid disease (AITD) (including Graves' disease (GD), Hashimoto's Thyroiditis (HT, Hashimoto's Thyroiditis)), myasthenia gravis (MG), insulin-dependent diabetes mellitus (IDDM), idiopathic thrombocytopenic purpura ( ITP), ankylosing spondylitis (Ankylosing spondylitis), Sjögren's syndrome (SS), autoimmune liver disease (AILD), acute disseminated (disseminated) encephalomyelitis (ADE, experimental Autoimmune encephalomyelitis (MOG).
  • AITD autoimmune Autoimmune thyroid disease
  • GD
  • the food and nutritional preparations include conventional formula food, formula food for special medical use, enteral nutrition preparation and parenteral nutrition preparation.
  • the conventional formula includes at least one of infant formula, pregnancy and lactation formula.
  • formulas for special medical purposes include premature/low birth weight infant formula, lactose-free or low-lactose formula, milk protein partial hydrolysis formula, milk protein deep hydrolysis formula or amino acid formula, breast milk and infant formula.
  • Food for Special Medical Purpose (FSMP) is a formula food specially processed and formulated to meet the special needs of people with food restriction, digestion and absorption disorders, metabolic disorders or specific disease states. Consume or combine with other foods.
  • Formula food for special medical purpose is a food for special dietary use. When the target population cannot eat ordinary diet or cannot meet their nutritional needs with daily diet, formula food for special medical purpose can be used as a way of nutritional supplementation for treatment, rehabilitation and maintenance of body functions. and other aspects play an important role in nutritional support.
  • the parenteral nutrition preparation includes at least one of fat emulsion injection, all-in-one nutrition solution and intravenous injection.
  • the enteral nutrition preparation includes at least one of amino acid type enteral nutrition preparation, short peptide type enteral nutrition preparation, whole protein type enteral nutrition preparation and component type enteral nutrition preparation.
  • folic acid or its derivatives in the present invention is not important, because the embodiment of the present invention has proved that the effect can be achieved by intraperitoneal injection of this composition, so adding folic acid or its derivatives to the formula suitable for infants and young children and formula milk powder for pregnant women, formula food for special medical purposes, and other enteral and parenteral nutritional preparations or pharmaceutical excipients.
  • folic acid or its derivatives to the formula suitable for infants and young children and formula milk powder for pregnant women, formula food for special medical purposes, and other enteral and parenteral nutritional preparations or pharmaceutical excipients.
  • the prevention and treatment of biliary atresia, liver fibrosis, cholangitis, and gastrointestinal bacteria can be achieved. Group disorders, inflammatory bowel disease, viral infections, neonatal infectious diseases and other diseases.
  • the neonatal infection also includes neonatal lung infection (such as neonatal pneumonia), neonatal umbilical cord infection, neonatal brain infection, neonatal skin infection, neonatal urinary system infection, neonatal oral infection , neonatal eye infection, neonatal blood infection (eg, neonatal sepsis), neonatal biliary tract infection, neonatal stomach infection, neonatal intestinal infection (eg, neonatal infection leading to gastrointestinal disease), neonatal liver infection (such as neonatal hepatitis syndrome), or concurrent infection of multiple parts of the newborn's body.
  • neonatal lung infection such as neonatal pneumonia
  • neonatal umbilical cord infection neonatal brain infection
  • neonatal skin infection neonatal skin infection
  • neonatal urinary system infection neonatal oral infection
  • neonatal eye infection neonatal blood infection (eg, neonatal sepsis), neonatal biliary tract infection
  • neonatal stomach infection eg, neonatal intestinal infection leading to gastrointestinal disease
  • hereditary diseases include biliary atresia
  • infectious diseases include cholangitis, Jaundice, neonatal infection, necrotizing enterocolitis, bacterial infectious disease, viral infectious disease, said allergic disease including food allergy.
  • the biliary atresia includes virus-induced biliary atresia, or cholestasis-induced biliary atresia.
  • the virus-induced biliary atresia also includes biliary atresia mainly caused by cytomegalovirus, or biliary atresia mainly caused by rotavirus.
  • the biliary atresia also manifests as food allergy caused by biliary atresia, jaundice caused by biliary atresia, cholangitis caused by biliary atresia, liver disease caused by biliary atresia, bowel disease caused by biliary atresia tract diseases.
  • the liver disease caused by biliary atresia includes liver function damage caused by biliary atresia, and/or liver inflammatory disease caused by biliary atresia.
  • the intestinal disease caused by biliary atresia includes intestinal inflammatory disease caused by biliary atresia.
  • the jaundice includes pathological jaundice or infantile jaundice.
  • the pathological jaundice includes pathological jaundice caused by a virus, or pathological jaundice in infancy.
  • the pathological jaundice also includes pathological jaundice in infancy caused by viruses; or the pathological jaundice caused by viruses includes cytomegalovirus jaundice.
  • the cholangitis includes bacterial cholangitis, viral cholangitis, cholangitis caused by biliary atresia, and cholangitis complicated by postoperative biliary atresia.
  • the bacterial cholangitis also includes bacterial cholangitis caused by biliary atresia and bacterial cholangitis complicated by postoperative biliary atresia.
  • the neonatal infectious disease also includes neonatal bacterial infection, neonatal viral infection, neonatal mycoplasma or neonatal chlamydia infection; or, the neonatal infectious disease Including neonatal hepatitis syndrome, neonatal sepsis, neonatal pneumonia, and gastrointestinal diseases caused by neonatal infection.
  • the neonatal virus infection also includes RNA virus and DNA virus.
  • the RNA virus also includes antisense RNA virus and DNA virus.
  • the neonatal virus infection also includes neonatal cytomegalovirus (CMV) infection, neonatal rotavirus infection, and neonatal herpes virus infection.
  • CMV neonatal cytomegalovirus
  • the bacterial infectious disease includes Escherichia coli infection, Staphylococcus aureus infection or hemolytic streptococcus infection.
  • the viral infectious disease includes cytomegalovirus (CMV) infection, rotavirus infection, and herpes virus infection.
  • CMV cytomegalovirus
  • the viral infectious disease further includes viral diarrhea.
  • the food allergy includes food allergy caused by or accompanied by biliary atresia, food allergy caused by or accompanied by choledochal cyst; or, the food allergy also manifests Skin diseases caused by food allergies, gastrointestinal diseases caused by food allergies, and respiratory diseases caused by food allergies.
  • the gastrointestinal disease caused by food allergy includes inflammatory bowel disease caused by food allergy.
  • the folic acid derivatives include pharmaceutically acceptable salts, esters, hydrates, solvates, polymorphs, tautomers, prodrugs and folic acid functional equivalent.
  • the folic acid derivative includes any one of (a1) to (a2):
  • (a2) is a pharmaceutically acceptable salt of (a1).
  • the pharmaceutical salt includes calcium, sodium, zinc, arginine, choline, acetylcholine, N-methylaminoethanol, 2-amino-2- Methyl-propanol, 1,1-dimethylbiguanide, phenethylbiguanide, diaminoguanidine, glucosamine, and dimethylaminoethanol.
  • the folic acid derivative includes calcium folinate.
  • the medicine further contains other active ingredients, and the other active ingredients include vitamin B, vitamin C, nicotinamide, zinc oxide, vitamin A, vitamin E, vitamin D, vitamin K, At least one of homocysteine, glutathione and taurine.
  • the vitamin B contains at least one of vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7 and vitamin B12.
  • the drug includes folic acid or its derivatives, and at least one of vitamin B6 and B12; or the drug includes: folic acid or its derivatives; and vitamin B6, vitamin B12 , vitamin A, vitamin E, vitamin C, nicotinamide, zinc oxide, taurine; or, the drug includes folic acid or its derivatives and nicotinamide; or, the drug includes folic acid or its derivatives substances and vitamin E.
  • the dosage form of the drug is a dosage form suitable for children or a dosage form suitable for adults.
  • the children include newborns within 28 days of birth, infants under 1 year old, infants aged 1 to 6 years, and children aged 6 to 18 years.
  • the adult includes a pregnant female adult, a perinatal female adult and a lactating female adult.
  • the dosage form of the drug includes a dosage form for gastrointestinal administration and a dosage form for parenteral administration.
  • the dosage forms of the drug include capsules, tablets, microcapsule preparations, injections, suppositories, sprays, powders, soft capsules, dripping pills, honeyed pills, pills, granules , honey refining paste, sustained and controlled release preparation, oral liquid preparation, injection, chewable tablet, oral tablet, transdermal patch and effervescent tablet.
  • the unit dose of the folic acid or its derivatives in the drug is 0.1 mg to 1 g.
  • the unit dose of the folic acid or its derivatives in the drug is 0.1mg, 0.2mg, 0.3mg, 0.4mg, 0.5mg, 0.6mg, 0.7mg, 0.8mg , 0.9mg or 1.0mg.
  • the unit dose of the folic acid or its derivatives in the medicament is 0.2 mg, 0.4 mg, 0.8 mg, or 1.0 mg.
  • a medicament comprising: folic acid or a derivative thereof; and other active ingredients comprising vitamin B, vitamin C, vitamin A, vitamin E, vitamin D, vitamin K, niacinamide, zinc oxide, At least one of homocysteine, glutathione and taurine.
  • (a2) is a pharmaceutically acceptable salt of (a1).
  • the pharmaceutical salt includes calcium, sodium, zinc, arginine, choline, acetylcholine, N-methylaminoethanol, 2-amino-2-methyl-propane alcohol, 1,1-dimethylbiguanide, phenethylbiguanide, diaminoguanidine, glucosamine, and dimethylaminoethanol.
  • the children include newborns within 28 days of birth, infants under 1 year old, infants 1 to 6 years old, and children 6 to 18 years old;
  • the drug according to the above-mentioned [36], wherein the dosage form of the drug includes a dosage form for gastrointestinal administration and a dosage form for parenteral administration.
  • the dosage form of the drug includes capsules, tablets, microcapsule preparations, injections, suppositories, sprays, powders, soft capsules, dripping pills, honeyed pills, pills, and granules , honey refining ointment, sustained and controlled release preparation, oral liquid preparation, injection, chewable tablet, oral tablet, transdermal patch and effervescent tablet.
  • a method for preventing and/or treating a hereditary, infectious or allergic disease comprising administering to a subject a drug comprising folic acid or a derivative thereof as an active ingredient, wherein the hereditary disease includes biliary atresia , the infectious disease includes cholangitis, jaundice, neonatal infection, necrotizing enterocolitis, bacterial infectious disease, viral infectious disease, and the allergic disease includes food allergy.
  • biliary atresia includes virus-induced biliary atresia, or cholestasis-induced biliary atresia.
  • the virus-induced biliary atresia also includes biliary atresia mainly caused by cytomegalovirus, or biliary atresia mainly caused by rotavirus.
  • the biliary atresia is also manifested as food allergy caused by biliary atresia, jaundice caused by biliary atresia, cholangitis caused by biliary atresia, liver disease caused by biliary atresia, bowel disease caused by biliary atresia tract disease.
  • the liver disease caused by biliary atresia includes liver function damage caused by biliary atresia, and/or liver inflammatory disease caused by biliary atresia.
  • the intestinal disease caused by biliary atresia includes intestinal inflammatory disease caused by biliary atresia.
  • the pathological jaundice further includes pathological jaundice in infancy caused by viruses; or preferably, the pathological jaundice caused by viruses includes cytomegalovirus jaundice.
  • the cholangitis includes bacterial cholangitis, viral cholangitis, cholangitis caused by biliary atresia, and cholangitis complicated by postoperative biliary atresia.
  • the bacterial cholangitis also includes bacterial cholangitis caused by biliary atresia and bacterial cholangitis complicated by postoperative biliary atresia.
  • the neonatal infectious disease also includes neonatal bacterial infection, neonatal viral infection, neonatal mycoplasma or neonatal chlamydia infection; or the neonatal infectious disease includes Neonatal hepatitis syndrome, neonatal sepsis, neonatal pneumonia, and gastrointestinal diseases caused by neonatal infection.
  • the neonatal virus infection further includes RNA virus and DNA virus.
  • the RNA virus further includes antisense RNA virus and DNA virus.
  • the neonatal viral infection also includes neonatal cytomegalovirus (CMV) infection, neonatal rotavirus infection, and neonatal herpes virus infection.
  • CMV neonatal cytomegalovirus
  • the food allergy is also manifested as skin disease caused by food allergy, gastrointestinal disease caused by food allergy, and respiratory disease caused by food allergy.
  • the gastrointestinal disease caused by food allergy includes inflammatory bowel disease caused by food allergy.
  • the folic acid derivatives include pharmaceutically acceptable salts, esters, hydrates, solvates, polymorphs, tautomers, prodrugs and folic acid functional equivalent.
  • (a2) is a pharmaceutically acceptable salt of (a1).
  • the pharmaceutical salt includes calcium, sodium, zinc, arginine, choline, acetylcholine, N-methylaminoethanol, 2-amino-2-methanol Dimethyl-propanol, 1,1-dimethylbiguanide, phenethylbiguanide, diaminoguanidine, glucosamine, and dimethylaminoethanol.
  • the treatment method further comprising administering to the subject other active ingredients, the other active ingredients comprising vitamin B, vitamin C, nicotinamide, zinc oxide, vitamin A, vitamin E, At least one of homocysteine, glutathione and taurine.
  • vitamin B contains at least one of vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7 and vitamin B12.
  • the drug includes folic acid, and at least one of vitamin B6 and B12; or, the drug includes: folic acid or a derivative thereof; and vitamin B6, vitamin B12, vitamin A , vitamin E, vitamin C, vitamin D, vitamin K, nicotinamide, zinc oxide, taurine; or, the drug includes folic acid or its derivatives, and nicotinamide; or, the drug includes folic acid or its derivatives, and vitamin E; or, the medicament comprises folic acid or its derivatives, and vitamin A and vitamin E.
  • the dosage form of the drug is a dosage form suitable for children or a dosage form suitable for adults.
  • the dosage form of the drug includes a dosage form for gastrointestinal administration and a dosage form for parenteral administration.
  • the dosage forms of the drug include capsules, tablets, microcapsule preparations, injections, suppositories, sprays, powders, soft capsules, dripping pills, honeyed pills, pills, and granules , honey refining ointment, sustained and controlled release preparation, oral liquid preparation, injection, chewable tablet, oral tablet, transdermal patch and effervescent tablet.
  • the unit dose of the folic acid or its derivatives in the drug is 0.1 mg to 1 g.
  • the unit dose of the folic acid or its derivatives in the drug is 0.1mg, 0.2mg, 0.3mg, 0.4mg, 0.5mg, 0.6mg, 0.7mg, 0.8mg , 0.9mg or 1.0mg.
  • the unit dose of the folic acid or its derivative in the medicament is 0.2 mg, 0.4 mg, 0.8 mg, or 1.0 mg.
  • Diagnostic or auxiliary diagnostic markers for biliary atresia comprising:
  • the marker according to [86], the S100a8, S100a9, Nox2 and IFN- ⁇ include proteins and/or mRNAs.
  • the tissue includes liver and intestinal tract.
  • the excrement includes feces and urine.
  • the markers include at least one of folic acid, S100a8, S100a9, Nox2, and IFN- ⁇ .
  • the S100a8, S100a9, Nox2, and IFN- ⁇ include proteins and/or mRNAs.
  • the product includes a reagent, a kit, a gene chip and a protein chip.
  • the product contains primers and/or detection probes for detecting the marker and/or internal reference mRNA .
  • the product when the marker is S100a8mRNA, the product includes primers for detecting S100a8mRNA, and the nucleotide sequences of the primers are shown in SEQ ID NO.1 and SEQ ID NO.2.
  • the product when the marker is S100a9mRNA, the product includes primers for detecting S100a9mRNA, and the nucleotide sequences of the primers are shown in SEQ ID NO.3 and SEQ ID NO.4.
  • the product when the marker is IFN- ⁇ FmRNA, the product includes primers for detecting IFN- ⁇ mRNA, and the nucleotide sequences of the primers are as shown in SEQ ID NO.5 and SEQ ID NO.6 Show.
  • the product when the marker is Nox2 mRNA, the product includes primers for detecting Nox2 mRNA, and the nucleotide sequences of the primers are shown in SEQ ID NO.7 and SEQ ID NO.8.
  • test object meets at least one of (c1)-(c5), it is diagnosed as a patient with biliary atresia disease;
  • the reference level is the level of subjects of the same age who do not suffer from biliary atresia disease.
  • the subject in the same age group is a healthy person or a choledochal cyst patient.
  • the marker is from body fluid, blood, tissue, cell or excrement.
  • the tissue includes liver and intestinal tract.
  • the excrement includes feces and urine.
  • the blood is whole blood, serum or plasma, or dried blood spots.
  • the subject to be tested is an adult or a child.
  • the children include newborns within 28 days of birth, infants under 1 year old, infants aged 1-6 years, and children aged 6-8 years.
  • the adult is selected from a pregnant female adult, a perinatal female adult, or a lactating female.
  • a kit comprising reagents for detecting markers; said markers include at least one of folic acid, S100a8, S100a9, Nox2, and IFN- ⁇ .
  • the product when the detection marker is the mRNA of S100a8, S100a9, Nox2 and IFN- ⁇ , the product contains primers for detecting the marker and/or internal reference mRNA, and/or detects probe.
  • the product when the detection markers are mRNAs of S100a8, S100a9, Nox2 and IFN- ⁇ , the product also includes: sample processing reagents, including but not limited to sample lysing reagents, sample purification Reagents and/or sample nucleic acid extraction reagents.
  • sample processing reagents including but not limited to sample lysing reagents, sample purification Reagents and/or sample nucleic acid extraction reagents.
  • the product when the detection markers are mRNAs of S100a8, S100a9, Nox2 and IFN- ⁇ , the product also includes: RNA extraction reagent, dNTP, DNA polymerase, double-strand specific fluorescent One or more of dyes and water.
  • the product when the detection marker is S100a8mRNA, the product includes primers for detecting S100a8mRNA, and the nucleotide sequences of the primers are shown in SEQ ID NO.1 and SEQ ID NO.2.
  • the product when the detection marker is S100a9mRNA, the product includes primers for detecting S100a9mRNA, and the nucleotide sequences of the primers are shown in SEQ ID NO.3 and SEQ ID NO.4.
  • the product when the detection marker is IFN- ⁇ mRNA, the product includes primers for detecting IFN- ⁇ mRNA, and the nucleotide sequences of the primers are as SEQ ID NO.5 and SEQ ID NO. 6.
  • the product when the detection marker is Nox2 mRNA, the product includes primers for detecting Nox2 mRNA, and the nucleotide sequences of the primers are as shown in SEQ ID NO.7 and SEQ ID NO.8 Show.
  • the detection sample or test sample of the product is from the body fluid, blood, tissue, cell or excrement of the subject; further preferably, the marker is from blood, tissue or excrement.
  • test subject is an adult or a child.
  • kits according to [125] wherein the adult is selected from a pregnant female adult, a perinatal female adult, or a lactating female adult.
  • test object meets at least one of (c1)-(c5), it is diagnosed as a patient with biliary atresia;
  • the reference level is the level of subjects of the same age who do not suffer from biliary atresia disease.
  • the subject in the same age group is a healthy person or a choledochal cyst patient.
  • a method for diagnosing or assisting in diagnosing biliary atresia comprising detecting markers: the level and/or activity of at least one of folic acid, S100a8, S100a9, Nox2, and IFN- ⁇ .
  • the detection sample or test sample of the method is from body fluid, blood, tissue, cell or excrement of the subject; further preferably, the marker is from blood, tissue or excrement.
  • the tissue includes liver, intestinal tract.
  • the excrement includes feces and urine.
  • the blood is whole blood, serum or plasma, or dried blood spots; further, serum or dried blood spots.
  • the subject to be tested is an adult or a child.
  • the children include newborns within 28 days of birth, infants under 1 year old, infants aged 1 to 6 years, and children aged 6 to 8 years.
  • the reference level is the level of subjects of the same age who do not suffer from biliary atresia disease.
  • the subject in the same age group is a healthy person or a choledochal cyst patient.
  • the detection method when the detection marker is the mRNA of S100a8, S100a9, Nox2 and IFN- ⁇ , the detection method further includes using the marker and/or internal reference mRNA primers, and/or Detection probe.
  • the detection method when the detection markers are mRNAs of S100a8, S100a9, Nox2 and IFN- ⁇ , the detection method further includes using: sample processing reagents, including but not limited to sample lysing reagents, sample Purification reagents and/or sample nucleic acid extraction reagents.
  • sample processing reagents including but not limited to sample lysing reagents, sample Purification reagents and/or sample nucleic acid extraction reagents.
  • the detection method when the detection markers are mRNAs of S100a8, S100a9, Nox2 and IFN- ⁇ , the detection method also includes using: RNA extraction reagent, dNTP, DNA polymerase, double-strand specificity One or more of fluorescent dyes and water.
  • the detection method when the detection marker is S100a8mRNA, the detection method also includes the use of S100a8mRNA primers, the nucleotide sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2 .
  • the detection method when the detection marker is S100a9mRNA, the detection method also includes using primers for detecting S100a9mRNA, the nucleotide sequences of the primers are as shown in SEQ ID NO.3 and SEQ ID NO.4 Show.
  • the detection method when the detection marker is IFN- ⁇ mRNA, the detection method also includes using primers for detecting IFN- ⁇ mRNA, the nucleotide sequences of the primers are as SEQ ID NO.5 and SEQ ID Shown in NO.6.
  • the detection method when the detection marker is Nox2 mRNA, the detection method also includes the use of primers for detecting Nox2 mRNA, the nucleotide sequences of the primers are as SEQ ID NO.7 and SEQ ID NO. 8.
  • the present invention uses a high security nutrient substance that people need every day - folic acid or its derivatives, to prevent and treat biliary atresia and its highly relevant disease cholangitis, folic acid or its derivatives (such as its medicine salt) as a safe water-soluble vitamin, can be directly applied, added to nutritional food or nutritional preparations for pregnancy, pregnancy, breastfeeding women and/or newborn babies, thereby fundamentally preventing the occurrence of biliary atresia, development and prognosis, and less toxic side effects.
  • the examples of the present invention have proved that supplementing folic acid can also inhibit the expression of intestinal inflammatory factors, reduce intestinal iron content, and correct intestinal flora disorders; thus, the present invention provides an economical, safe and effective
  • the composition for regulating the balance of intestinal flora and preventing and treating intestinal inflammatory diseases such as inflammatory bowel disease, whose active ingredient is folic acid or its derivatives, provides another method for preventing and treating acute/chronic, Feasible solutions for inflammatory/non-inflammatory diseases.
  • the examples of the present invention show that the use of folic acid in the virus-infected biliary atresia mouse model can significantly reduce the jaundice rate of mice, improve the liver function of mice, and inhibit the expression of liver or intestinal cytotoxic cytokine IFN- ⁇ , At the same time, it can also inhibit the expression of intestinal inflammatory factors, reduce the content of intestinal iron, and correct the disorder of intestinal flora. Therefore, the present invention provides a safe active ingredient that can act on the gallbladder, liver, and intestinal tract at the same time—folic acid.
  • the present invention can also use its target Diversity, to achieve the prevention and treatment of diseases such as liver fibrosis, enterohepatic circulation disorder, virus infection, and relatively mild disease of biliary atresia - cholangitis.
  • folic acid can inhibit the expression of inflammatory cytokines S100a8, S100a9 and IFN- ⁇ in the intestinal tract and/or liver, as well as the iron ion overload situation that occurs in the disease, and promote the
  • Nox2 a cell-specific transcription factor, was derived from the source, and it was proved that at least one of folic acid, S100a8, S100a9, IFN- ⁇ , and Nox2 can be used as a marker for the diagnosis or auxiliary diagnosis of biliary atresia.
  • the present invention also proves that folic acid can also reduce the total bilirubin concentration and direct bilirubin concentration in patients with biliary atresia, so as to achieve the effect of preventing and/or treating cholangitis.
  • folic acid supplementation can also correct systemic iron overload and luminal iron deficiency, inhibit eosinophil infiltration in the small intestine, inhibit the production of IgG-Ro/SSA autoantibodies, increase the ratio of IgM/IgG4, and at the same time, prolong the period of autoimmunity and improve Liver function;
  • the present invention also proves that supplementing calcium folinate (CF) can reduce the content of INF ⁇ and ROS in the liver, reduce the incidence of jaundice and improve neonatal growth; increase the expression of Dhfr, Mthfr, Nrf2, Slc40a1 in the small intestine , so as to restore folic acid metabolism, oxidative stress and iron homeostasis;
  • nutritional food and/or preparations and/or drugs added with folic acid or its derivatives are a safe, effective, economical and feasible way to prevent and treat biliary atresia, cholangitis, liver fibrosis, enterohepatic circulation disorder, inflammatory bowel disease, Viral infections, regulation of intestinal flora balance, methods of infection of newborns.
  • Fig. 1 is the result graph of folic acid content in patients with biliary atresia (BA) and choledochal cyst (CC): wherein, A is the result graph of folic acid level in serum of patients with biliary atresia (BA) and choledochal cyst (CC); Receiver operating characteristic (ROC) curve plot of predictive performance for differentiating BA from non-BA subjects (choledochal cyst (CC) patients).
  • BA biliary atresia
  • CC choledochal cyst
  • Fig. 2 is the result figure of the prevention and treatment effect of folic acid on the mouse model of biliary atresia: wherein, A is a schematic diagram of the treatment scheme of BA model mice; B is the result figure of folic acid on the weight of the mouse model of biliary atresia; C is folic acid The result graph of the effect on the jaundice rate of the biliary atresia mouse model; D is the result graph of the effect of folic acid on the liver necrosis and lymphocyte infiltration of the biliary atresia mouse model; E is the effect of folic acid on the CD4 + in the liver of the biliary atresia mouse model The result graph of the effect of IFN- ⁇ content in T cells.
  • Fig. 3 is a graph showing the effect of folic acid on the intestinal epithelium of the mouse model of biliary atresia.
  • Figure 4 is a graph showing the effect of folic acid on the intestinal villi of a mouse model of biliary atresia.
  • Fig. 5 is a graph showing the effect of folic acid on inflammatory factors in a mouse model of biliary atresia.
  • Figure 6 is a graph showing the effect of folic acid on the overactivation of the type I interferon pathway caused by viral or bacterial infection.
  • Figure 7 is the result graph of the prevention and treatment effect of calcium folinate on the mouse model of biliary atresia: wherein, A is the result graph of the effect of calcium folinate on the body weight of the mouse model of biliary atresia; B is the effect of calcium folinate on the mouse model of biliary atresia The result graph of the influence of lymphocyte infiltration around the liver portal area, the structural integrity of the liver lobule, and the ballooning degeneration of intestinal epithelial cells in the model; C is the result graph of the effect of calcium folinate on the jaundice rate of the biliary atresia mouse model; D is the result graph of the effect of calcium folinate on the content of INF ⁇ and ROS in the liver of the mouse model of biliary atresia; E is the effect of calcium folinate on the expression of Dhfr, Mthfr, Nrf2, and Slc40a1 in the small intestine
  • Figure 8 is a diagram of the effect of folic acid on the incidence of cholangitis in infants with biliary atresia: where, A is a schematic diagram of the treatment plan for infants with BA; B is the demographic and clinical characteristics of infants with BA; C is the effect of folic acid on cholangitis in infants with biliary atresia Summary plot of the effect on morbidity.
  • Figure 9 is a diagram of the effect of folic acid on the incidence of cholangitis in infants with biliary atresia: where, A is the incidence of cholangitis in different time periods after infants with biliary atresia receive folic acid treatment; B is the influence of folic acid on the concentration of total bilirubin in infants with biliary atresia ; C is the effect of folic acid on the direct bilirubin concentration in infants with biliary atresia.
  • Figure 10 is the results of research on the mechanism of folic acid treatment of biliary atresia: A is a schematic diagram of the design of single-cell sequencing experiments based on the BD Rhapsody "M" platform; B is the effect of folic acid on the distribution and proportion of intestinal cells; C is folic acid Graphical summary of the mechanisms of treatment of biliary atresia.
  • Figure 11 is a diagram of the influence of folic acid on the gene expression of enterocytes and the concentration of BH4 in plasma and HCY in liver biopsy: among them, A is the influence diagram of folic acid on the expression of indicator genes in enterocyte subsets; B is the influence of folic acid on the expression of enterocytes The influence diagram of SLC11A2, SLC40A1 and SLC46A1 gene expression; C is the influence diagram of folic acid on the concentration of BH4 in plasma and HCY in liver biopsy.
  • Figure 12 is a diagram of the influence of folic acid on iron concentration and the ratio of Ro/SSA IgG, Ro/SSA IgM, and IgM/IgG in the liver: where, A is the influence diagram of folic acid on iron concentration; B is the effect of folic acid on Ro/SSA IgG in the liver , Ro/SSA IgM, IgM/IgG ratio influence diagram.
  • Figure 13 is the result of folic acid inhibiting the occurrence of food allergy in patients with biliary atresia and cholecystoma: A is the survey result of food allergy after biliary atresia and cholecystectomy; A graph showing a significant reduction in the number of eosinophils involved in food allergies in the gut of a patient.
  • the medicine or composition should contain at least 0.1 mg of folic acid.
  • the requirement of folic acid for normal people is 0.2-0.4mg/day, and that for pregnant women and breast milk is 0.4mg/day.
  • a higher amount of folic acid is required, for example, 0.8mg-1g/day.
  • the drug is preferably presented as a unit dosage, ie a preparation of the active ingredient required for a single administration. Common unit preparations such as a unit (tablet) tablet, a unit (needle) injection or powder injection, etc., wherein the active ingredient is the amount required for one administration.
  • the amount required for one administration can be conveniently calculated by calculating the product of the body weight of the subject and the dose per unit body weight required for one administration of the subject (hereinafter referred to as "dose").
  • dose the dose per unit body weight required for one administration of the subject
  • the body weight of an adult is usually determined as 60 kg, and this body weight value can be used for calculation.
  • the dose per body weight of different subjects can be calculated through the equivalent dose conversion relationship.
  • equivalent dose conversion relationship between experimental animals and people known to those of ordinary skill in the art usually refer to the guidance of drug regulatory agencies such as FDA and SFDA, and also refer to "Huang Jihan et al. Equivalent dose conversion between animals and humans.
  • the active ingredient folic acid of the present invention or its derivative can be given daily dosage 0.8mg ⁇ 1g/day to the patient, and dosage concrete can be according to factors such as patient's gender, body weight, age, the degree of symptom actual. The situation is adjusted.
  • folic acid is folic acid (PTEROYLMONOGLUTAMATE), or including all its functional equivalents, and its derivatives, one or more leaf-based polyglutamic acid salts, wherein folic acid or compounds in which the pyrazine ring of the pterin moiety of polyglutamic acid is reduced to dihydrofolate or tetrahydrofolate, or derivatives of all of the above compounds, where the N-5 or N-10 position is carried at various oxidation levels A carbon unit, or a pharmaceutically compatible salt thereof, or a combination of two or more thereof.
  • folic acid which exists as free folic acid (pteroylglutamate) or folinic acid (leucovorin).
  • folic acid equivalents or derivatives are optional: folinic acid, dihydrofolate, tetrahydrofolate, 5-methyltetrahydrofolate, 5,10-methylenetetrahydrofolate, 5,10-methylenetetrahydrofolate Folic acid, 5,10-formyltetrahydrofolate, 5-formyltetrahydrofolate (Cryptophyllin), 10-formyltetrahydrofolate, 10-methyltetrahydrofolate, their pharmaceutically acceptable salts , or a combination of two or more thereof.
  • vitamin B group includes vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), and vitamin B2 (riboflavin). ), vitamin B6 (pyridoxine), vitamin B12 (cyanocobalamin), vitamin B7 (biotin), etc., are effective ingredients to promote the absorption of folic acid.
  • vitamin C has the effect of enhancing the curative effect of folic acid.
  • vitamin A is a fat-soluble vitamin, an organic compound with a chemical formula of C 20 H 30 O, stable to heat, acid, and alkali, and easily oxidized, and ultraviolet rays can promote its oxidative destruction.
  • Vitamin A includes A1 and A2, and A1 is retinol.
  • Vitamin A2 is 3-dehydroretinol.
  • Vitamin A has various physiological functions such as promoting growth and reproduction, maintaining bone, epithelial tissue, vision and normal secretion of mucosal epithelium, and vitamin A and its analogues can prevent precancerous lesions.
  • Homocysteine (also known as homocysteine, plasma homocysteine, homocysteine, Hcy) is an intermediate product in the process of methionine metabolism, and its structural formula is HSCH 2 (NH 2 )CO 2 H.
  • HSCH 2 (NH 2 )CO 2 H There are two forms of oxidized and reduced HCY in plasma.
  • the oxidized form contains disulfide groups, including homocysteine and cystine; the reduced form contains sulfur groups, including homocysteine and cysteine.
  • the "taurine” mentioned in the present invention has liver protection and liver protection effects, can promote gastrointestinal function, increase human immunity, improve body resistance, and improve disease resistance.
  • Those skilled in the art should understand that the Its application in the treatment of biliary atresia can shorten the course of treatment of biliary atresia with liver damage.
  • biliary atresia (biliary atresia, BA) is one of the common serious hepatobiliary system diseases in infancy, characterized by progressive inflammation and fibrosis of intrahepatic and external bile ducts. If it is not treated in time, biliary cirrhosis, portal hypertension, and liver failure will occur in the late stage. It is an occlusive lesion of unknown etiology involving the intrahepatic and extrahepatic bile ducts, leading to cholestasis and progressive liver fibrosis until cirrhosis and life-threatening diseases.
  • enterohepatic circulation disorder For the "enterohepatic circulation disorder” mentioned in the present invention, those skilled in the art should understand that the diseases caused by the enterohepatic circulation disorder include hypercholesterolemia, cholesterol gallstones, and jaundice.
  • inflammatory bowel disease is an idiopathic intestinal inflammatory disease involving the ileum, rectum, and colon. Clinical Diarrhea, abdominal pain, and even bloody stool. This disease includes ulcerative colitis (UC) and Crohn's disease (CD). Ulcerative colitis is a continuous inflammation of the colonic mucosa and submucosa. The disease usually involves the rectum first. Gradually spread to the whole colon, Crohn's disease can involve the whole digestive tract, which is non-continuous full-thickness inflammation, and the most commonly involved parts are the terminal ileum, colon and perianal area.
  • IBD inflammatory bowel disease
  • IBD inflammatory bowel disease
  • Ulcerative colitis is a continuous inflammation of the colonic mucosa and submucosa. The disease usually involves the rectum first. Gradually spread to the whole colon, Crohn's disease can involve the whole digestive tract, which is non-continuous full-thickness inflammation, and the most commonly involved parts are the terminal
  • viral infection refers to the process in which viruses invade the body through various ways and multiply in susceptible host cells.
  • Human viruses refer to Viruses that can infect the human body or have pathogenic effects on humans. The essence of viral infection is the process of interaction between the virus and the body, and between the virus and susceptible cells. Virus infection often produces varying degrees of damage or virus due to different virus types and body states. Diseases. Viruses cause disease by invading the host and infecting cells, and the pathogenic effect is manifested in two aspects of the human body and cells.
  • intestinal flora disorder For the intestinal flora disorder mentioned in the present invention, those skilled in the art should understand that there will be a stable and balanced flora in the intestinal tract of healthy people to maintain the normal intestinal environment, once this intestinal environment is destroyed It will cause intestinal flora disorder, and this disease will inevitably cause diseases of the body's digestive system. Diseases easily caused by intestinal flora disorders include but are not limited to:
  • antibiotics tetracyclines, ampicillin, etc.
  • adrenal cortex hormones and intestinal surgery in elderly patients or patients with chronic diseases.
  • Betamycin produced by Clostridium perfringens can cause acute necrotic tumors, wasting diseases, and infections are most likely to occur under the use of antibiotics and corticosteroids.
  • NEC Newcastle disease virus
  • necrosis of the mucosa and even the deep layers of the intestine most often in the terminal ileum
  • the colon and proximal small intestine are rarely involved, and epidemiological studies have confirmed that some sporadic cases are associated with the colonization of special pathogens in the intestine (such as Klebsiella, Escherichia coli, coagulase-negative staphylococci).
  • Intestinal necrosis starts from the mucosal layer and gradually involves the entire thickness of the intestinal wall, resulting in perforation and secondary sepsis in 1/3 of newborns.
  • Pseudomonas aeruginosa is an opportunistic pathogenic bacterium, which is often a secondary infection. Infection is most likely to occur in infants, the elderly, certain malignant tumors, wasting diseases, and the use of antibiotics and corticosteroids.
  • Proteus can be conditional pathogenic bacteria under certain conditions, such as common bacillus, mirabilis, morganii proteus can cause food poisoning, non-constant proteus can cause summer diarrhea in infants and young children.
  • the pneumonia bacteria normally parasitic in the intestinal tract can cause infection, especially severe diarrhea in children.
  • intestinal flora disorder It is very common for intestinal flora disorder to cause intestinal inflammation. At the same time, diarrhea is also the most important clinical symptom of intestinal flora disorder. If the symptoms of diarrhea are severe, it will also cause symptoms such as dehydration and blood pressure drop. If it is not treated in time, it may also cause diseases of the respiratory system or urinary system.
  • the gestational period refers to the physiological period from conception to delivery, also known as the pregnancy period. It is generally about 266 days from fertilization of mature eggs to delivery of the fetus. For ease of calculation, pregnancy is usually calculated from the first day of the last menstrual period, and full-term pregnancy is about 280 days (40 weeks). During pregnancy, the maternal metabolism, digestive system, respiratory system, vascular system, nervous system, endocrine system, Corresponding changes occur in the reproductive system, bone joint ligaments and breasts. The whole process of pregnancy is divided into three periods: before the 13th week of pregnancy, it is called early pregnancy; the 14th to 27th week is called second trimester pregnancy; the 28th week and thereafter are called late pregnancy pregnancy.
  • perinatal period refers to the period before and after delivery from 28 weeks of pregnancy to one week after delivery.
  • lactation period refers to the period during which a postpartum mother feeds her baby with her own milk, that is, the period from the beginning of breastfeeding to the end of breastfeeding, generally about 10 months to about 2 years.
  • biomarkers refer to biochemical indicators that can mark changes or possible changes in system, organ, tissue, cell and subcellular structure or function, with Very wide range of uses. Biomarkers can be used for disease diagnosis, judgment of disease stage, or to evaluate the safety and effectiveness of new drugs or new treatments in the target population. Those skilled in the art should understand that for disease research, biomarkers generally refer to certain characteristic biochemical indicators in a common physiological or pathological or therapeutic process that can be objectively measured and evaluated. The progress in the current biological process. Examining a disease-specific biomarker may help in disease identification, early diagnosis and prevention, and monitoring during treatment.
  • gamma interferon also known as gamma-IFN or immune interferon
  • gamma interferon is produced by mitogen-stimulated T lymphocytes.
  • Interferon is a highly effective antiviral biologically active substance and a lymphokine with extensive immune regulation.
  • Dosage forms for injection such as various powder injections and water injections.
  • Dosage form for respiratory administration such as isoproterenol hydrochloride aerosol.
  • Dosage form for skin administration such as boric acid lotion.
  • Dosage form for mucosal administration such as erythromycin eye ointment.
  • Dosage forms for cavity administration such as various suppositories for rectum, vagina, and urethra.
  • Liquid dosage form gas dosage form, solid dosage form and semi-solid dosage form.
  • excipients refer to excipients and additives used in biological drugs and dispensing prescriptions, that is, the general term for all materials except the main active ingredients.
  • excipients are often added in addition to the main drug and solvent to increase solubility and stability.
  • pharmaceutical excipients also have important functions such as solubilization, solubility enhancement, and sustained and controlled release. They are important components that may affect the quality, safety, and effectiveness of drugs.
  • the essence of the pharmaceutical preparation formulation design process is the process of screening and applying pharmaceutical excipients based on the characteristics of the drug and the requirements of the dosage form.
  • compositions are the basic materials and important components of pharmaceutical preparations, the material basis to ensure the production and development of pharmaceutical preparations, and play a key role in the formulation and production of pharmaceutical preparations. It not only gives the drug a certain dosage form. Moreover, it has a great relationship with improving the efficacy of drugs and reducing adverse reactions. Its quality reliability and diversity are the basis for ensuring the advancement of dosage forms and preparations.
  • solvents for example, it can be divided into solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants , flavoring agent, preservative, suspending agent, coating material, aromatic agent, anti-adhesive agent, integrating agent, penetration enhancer, pH regulator, buffering agent, plasticizer, surfactant, foaming agent, Defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, etc.
  • S100a8 and S100a9 encode S100A8 (S100 Calcium Binding Protein A8) and S100A9 (S100 Calcium Binding Protein A9) calcium binding proteins, often forming S100A8/ A9 heterodimer.
  • S100A8 and S100A9 are closely related to many pathological processes of chronic inflammatory diseases and various tumors.
  • S100A8 and S100A9 proteins mainly play an important regulatory role in inflammation by binding and activating Toll-like receptors and glycation end product receptors to mediate intracellular inflammatory signal transduction and other pathways.
  • the myeloid derived suppressive cells (MDSC) mentioned in the present invention are a group of innate immune cells with significant inhibitory function produced under pathological conditions (such as tumors, autoimmune diseases, infections, etc.), and their differentiation Abnormalities occur, leading to the accumulation of MDSCs, the regulation of multiple signaling pathways and the impact of various cytokines, Nox2 is one of them.
  • Nox2 means that Nox2 encodes NADPH oxidase 2, which is an important member of the NADPH oxidase family and plays an important regulatory role in inflammation.
  • genes/proteins described in the present invention those skilled in the art know that the expressions of genes and proteins are corresponding, therefore, when it is proved that the gene has a function, it also shows that the corresponding protein also has the corresponding function; vice versa Of course.
  • children refer to anyone under the age of 18, preferably, they are newborns within 28 days of birth, infants within 1 year old, children aged 1-6 Toddlers, and children over 6 years old and under 18 years old.
  • an adult refers to anyone over the age of 18.
  • the adult is a pregnant female adult, a perinatal female adult or a breastfeeding adult. period female adults.
  • the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.
  • Example 1 Acquisition of specimens from patients with biliary atresia
  • the present invention collects clinical intestinal tract, liver and blood samples, and detects metabolic related indicators.
  • Exclusion criteria (1) children with systemic inflammatory response syndrome or multisystem malformations; (2) children with unclear primary disease diagnosis; (3) children whose parents refused to participate in the study or could not obtain parental authorization; ( 4) Patients who did not undergo Kasai surgery; (5) Patients who could not be followed up in time; (6) Patients with pernicious anemia and suspected vitamin B12 deficiency.
  • Exit criteria Exit implemented by the researcher: (1) The subject has abnormal function of vital organs during the trial; (2) The subject has a serious adverse drug allergic reaction during the trial; (3) The subject is in the trial Poor compliance in the process; (4) The subject's condition worsens or serious adverse reactions need to stop the test drug or take other treatment methods during the test.
  • peripheral blood 0.5-1 mL of peripheral blood was collected from the patient at admission, during the operation, and after the operation, collected with a purple-tipped EDTA anticoagulant tube, and then placed in an ice box and transported to the laboratory for processing. After the laboratory received the specimens, 0.3 mL of whole blood was first collected and stored in a -80°C refrigerator, and the remaining blood was centrifuged (1800 rpm for 5 min) to collect serum and stored in a freezer.
  • the remaining blood cells were added to peripheral blood lymphocyte separation medium (2mL) or directly added to erythrocyte lysate (4mL) for separation or lysing to obtain red blood cell-free mononuclear cells (PBMC), for cell counting, freezing or performing the next step of flow cytometry Cytological staining.
  • peripheral blood lymphocyte separation medium (2mL) or directly added to erythrocyte lysate (4mL) for separation or lysing to obtain red blood cell-free mononuclear cells (PBMC), for cell counting, freezing or performing the next step of flow cytometry Cytological staining.
  • PBMC red blood cell-free mononuclear cells
  • the liver specimens obtained during the operation were sent for pathologically fixed sections, and the remaining part was placed in tissue preservation solution and transported to the laboratory in an ice box. After the laboratory received the specimens, the specimens were divided into two parts for processing, one part was directly frozen in a -80°C refrigerator after removing the blood on the surface of the tissue; Pellets were stained for flow cytometry after removal of hepatocytes.
  • embryo intestinal specimens After signing the consent form for voluntary termination of pregnancy and the informed consent for biomedical research, the Department of Obstetrics and Gynecology assists in obtaining embryo specimens. The placenta was stripped in vitro, the umbilical cord was cut, the abdominal cavity was opened, and the intestinal tract was taken out, fixed on ice with 4% paraformaldehyde for 2 hours, then transferred to 30% sucrose for overnight fixation, embedded and fixed with OCT the next day, and frozen for sectioning.
  • Serum folic acid (Serum Folic Acid) detection :
  • chromogenic reagent A 50 ⁇ L of chromogenic reagent A to each well, then add 50 ⁇ L of chromogenic reagent B, shake and mix gently, and develop color at 37°C in the dark for 15 minutes.
  • 50 ⁇ L of stop solution 50 ⁇ L to each well to terminate the reaction (the blue color turns yellow immediately).
  • ROC curve receiver operating characteristic, receiver operating characteristic
  • Infect rhesus monkey rotavirus within 24 hours of birth to establish a BA model.
  • RRV Infect rhesus monkey rotavirus
  • B in Fig. 2 is a diagram of the body weight of the mice over time. It can be seen from B in Fig. 2 that from the 11th day of modeling, the RRV-induced BA mouse model group was smaller than the control mouse group and the folic acid supplementation group. The body weight of the mice began to show significant differences (p ⁇ 0.01), especially on the 13th day, the significant difference in body weight between the RRV-induced BA mouse model group and the control mouse group and the folic acid supplementation group reached 0.001, while the control group There was no significant difference in body weight between the rat group and the folic acid supplemented group (p>0.05). Thus, folic acid supplementation significantly improved the weight loss of BA model mice induced by RRV infection.
  • C in Fig. 2 is the graph of the jaundice rate of mice changing with time, as can be seen from C in Fig. 2, at the 13th day when the experiment was terminated, the jaundice rate of the control mouse group was 0%, and RRV induced BA mouse model
  • D in Figure 2 is a representation of necrotic foci or lymphocytes in the mouse liver. It can be seen from D in Figure 2 that on the 13th day after the termination of the experiment, RRV induced necrotic foci and lymphocyte infiltration in the BA mouse model group Severe, but necrosis and lymphocyte infiltration in the folic acid supplementation group mice liver decreased, and the necrosis and lymphocyte infiltration in the control mice were close to, less obvious. It can be seen that folic acid exhibits a significant anti-inflammatory therapeutic effect in BA disease.
  • E in Figure 2 is the IFN- ⁇ content in CD4 + T cells in the liver. It can be seen from E in Figure 2 that on the 13th day after the end of the experiment, the RRV-induced BA mouse model group was significantly different from the control mouse group, folic acid The IFN- ⁇ content in CD4 + T cells in the liver showed a significant difference (p ⁇ 0.01) between the supplemented groups, while the IFN- ⁇ content in CD4 + T cells between the control mice group and the folic acid supplemented group did not Significant difference (p>0.05).
  • H&E staining Intestinal hematoxylin and eosin (H&E) staining (or HE staining for short).
  • the specific method of HE staining is as follows: animal tissues were fixed in 4% paraformaldehyde (PFA) for 24 hours, immersed in 50%, 60%, 70%, 80%, 90% and 100% ethanol for dehydration, and xylene was transparent twice. , embedded in paraffin, and made into 4 ⁇ m sections. After the slices were baked at 60°C, the paraffin sections were dewaxed twice in xylene for 15 minutes each time, rehydrated in 100% and 70% ethanol twice, and soaked in distilled water twice.
  • PFA paraformaldehyde
  • Hematoxylin was stained at room temperature for 6 minutes, and the floating color was washed away with running water. 1% hydrochloric acid alcohol color separation for 3s, placed in tap water, and the running water turned blue for 5min. Stain with eosin for 3 minutes, dehydrate with 70% and 100% ethanol twice, clear with xylene twice, seal with neutral resin and examine under microscope.
  • the above results indicate that folic acid has a significant therapeutic effect on BA mice by inhibiting the expression of toxic cytokines and repairing the intestinal barrier. Furthermore, it can be seen from B and C in Figure 2 that the RRV-induced biliary atresia model has obvious symptoms at about 7 days, and this experiment has administered folic acid at the same time as the first day of modeling, so the above results not only prove that folic acid has The therapeutic effect of BA disease, also proved the preventive effect of folic acid on BA. At the same time, in the follow-up study, the inventor further proved the preventive effect of folic acid on BA by pre-administering folic acid and setting a blank control, and then conducting RRV virus infection to establish a BA model.
  • Example 4 S100a8, S100a9, Nox2, IFN- ⁇ are used in the diagnosis of biliary atresia
  • RNA was extracted from the intestines of the mice in the control mouse group in Example 3; the RRV-induced BA mouse model group; and the folic acid supplementation group, and the RNA was extracted using Trizol one-step method.
  • the first strand of cDNA was synthesized using Promega reverse transcription kit. Primers were designed according to the principle of crossing introns to avoid genome contamination interference. The primer sequences are as follows:
  • S100a8 F:AAATCACCATGCCCTCTACAAG (SEQ ID NO.1); R:CCCACTTTTATCACCATCGCAA (SEQ ID NO.2);
  • S100a9 F:ATACTCTAGGAAGGAAGGACACC (SEQ ID NO.3); R:TCCATGATGTCATTTATGAGGGC (SEQ ID NO.4);
  • IFN- ⁇ F: CCGAACTGTACGGACATCACA (SEQ ID NO.5); R: TCATAGTGTTGAAATGGCTCCAG (SEQ ID NO.6);
  • Nox2 F: TTCCCCAATTCAATGTCTGG (SEQ ID NO.7); R: TCCACCTGACACGACTTGAGA (SEQ ID NO.8).
  • the S100a8 gene (and its corresponding protein) can be used as one of the effective markers for the diagnosis or auxiliary diagnosis of biliary atresia disease, and when the S100a8 gene or its protein is detected compared with those without biliary atresia disease
  • the levels of the corresponding indicators in subjects of the same age group are highly expressed, it can be considered that the individual from which the sample comes from suffers from biliary atresia or has a higher possibility of suffering from biliary atresia.
  • the gene S100a9 expression graph in Figure 5 in the RRV-induced BA mouse model group, the gene S100a9 was abnormally highly expressed, which was significantly different from the blank control group (p ⁇ 0.05); although its expression was high in the folic acid supplementation group Compared with the blank control group, but compared with the RRV-induced BA mouse model group, the effect of effectively reducing the expression of gene S100a9 was still achieved.
  • the S100a9 gene (and its corresponding protein) can be used as one of the effective markers for the diagnosis or auxiliary diagnosis of biliary atresia disease, and when the S100a9 gene or its protein is detected compared with those without biliary atresia disease
  • the levels of the corresponding indicators in subjects of the same age group are highly expressed, it can be considered that the individual from which the sample comes from suffers from biliary atresia or has a higher possibility of suffering from biliary atresia.
  • the gene IFN- ⁇ expression graph in Figure 5 in the RRV-induced BA mouse model group, the gene IFN- ⁇ expression was abnormally high, and in the folic acid supplementation group was only slightly higher than that in the blank control group, the gene IFN- ⁇ The expression in the RRV-induced BA mouse model group was significantly different from that in the folic acid supplemented group and the blank control group (p ⁇ 0.05), while there was no significant difference in the expression of the gene IFN- ⁇ in the folic acid supplemented group and the blank control group.
  • the IFN- ⁇ gene (and should be understood as the corresponding protein) can be used as one of the effective markers for the diagnosis or auxiliary diagnosis of biliary atresia disease, and when the IFN- ⁇ gene or its protein is detected
  • the levels of the corresponding indicators in subjects of the same age group with biliary atresia are highly expressed, it can be considered that the individual from which the sample comes has biliary atresia or has a higher possibility of suffering from biliary atresia.
  • the gene Nox2 expression was abnormally low, which was significantly different from the blank control group (p ⁇ 0.05); although its expression was low in the folic acid supplementation group Compared with the blank control group, but compared with the RRV-induced BA mouse model group, the effect of effectively increasing the expression of gene Nox2 was still achieved.
  • the Nox2 gene (and should be understood as the corresponding protein) can be used as one of the effective markers for the diagnosis or auxiliary diagnosis of biliary atresia disease, and when the Nox2 gene or its protein is detected relative to those without biliary atresia disease
  • the level of corresponding indicators in subjects of the same age group is low, it can be considered that the individual from which the sample comes from suffers from biliary atresia or has a higher possibility of suffering from biliary atresia.
  • S100a8, S100a9, IFN- ⁇ , and Nox2 are significantly different from those in the blank control group. Therefore, S100a8, S100a9, IFN- ⁇ , and/or Nox2 can be used as a diagnostic or diagnostic tool for biliary atresia.
  • supplementation of folic acid can reduce intestinal inflammation in BA model mice, thereby achieving the purpose of preventing intestinal diseases caused by biliary atresia.
  • Example 2 On the basis of Example 2, the inventors found through experiments that using folic acid in serum, combined with one or more of S100a8, S100a9, IFN- ⁇ , and Nox2 in intestinal tissue, can be used more accurately for biliary tract.
  • the diagnosis or auxiliary diagnosis of atresia showed higher accuracy, specificity, and higher AUC value.
  • Example 5 Preventive and therapeutic effects of calcium folinate on a mouse model of biliary atresia
  • mice (3) Observe the survival status of the mice: including the incidence of jaundice, growth weight, and liver and intestine manifestations of the mice.
  • Infect rhesus monkey rotavirus within 24 hours of birth to establish a BA model.
  • RRV Infect rhesus monkey rotavirus
  • the leucovorin was treated with calcium folinate, wherein the dose of calcium folinate was 5 mg/kg, injected intraperitoneally, 16 hours after virus infection, once a day, and the experiment was terminated on the 12th day.
  • the leucovorin supplementation group significantly reduced the lymphocyte infiltration around the hepatic portal area of the BA model mice; the leucovorin supplementation group significantly increased the structural integrity of the hepatic lobules of the BA model mice; Calcium folate supplementation group restored the ballooning degeneration of intestinal epithelial cells in BA model mice (B in Figure 7);
  • the jaundice rate of the control mouse group was 0%
  • the jaundice rate of the RRV-induced BA mouse model group was 100%
  • the jaundice rate of the mice in the leucovorin supplemented group was less than 70%
  • the jaundice rate of RRV-induced BA mouse model group and the jaundice rate of the mice in the calcium folinate supplemented group were significantly different (p ⁇ 0.05). It can be seen that the BA model mice were successfully modeled, while the calcium folinate supplemented group significantly reduced the The jaundice rate of the BA model mice, the jaundice rate was reduced from 100% of the RRV group to less than 70%, showing a significant therapeutic effect (C in Figure 7);
  • the IFN- ⁇ levels in the liver of CD4 + T cells and CD8 + T cells in the RRV-induced BA mouse model group and the control mouse group and the folic acid supplemented group were significantly different (p ⁇ 0.001), folinic acid calcium supplementation group significantly reduced the IFN- ⁇ content in CD4 + T cells and CD8 + T cells in the liver of BA model mice;
  • the IgG1 content in plasma cells in the liver between the supplementation groups showed a significant difference (p ⁇ 0.001), and the calcium folinate supplementation group significantly reduced the IgG1 content in the plasma cells in the liver of BA model mice (Fig. 7 F);
  • calcium folinate (CF) supplementation can reduce the content of INF ⁇ and ROS in the liver, reduce the incidence of jaundice and improve the growth of newborns; increase the expression of Dhfr, Mthfr, Nrf2, and Slc40a1 in the small intestine, so that folic acid metabolism, oxidation Restoration of stress and iron homeostasis; decreased IFN- ⁇ levels in CD4 + T cells, CD8 + T cells, and IgG1 levels in plasma cells in the liver; decreased lymphocyte infiltration around the hepatic portal area; increased structural integrity of the hepatic lobule ; The ballooning degeneration of intestinal epithelial cells can be recovered, and the effect of treating biliary atresia can be achieved.
  • CF calcium folinate
  • the inventors also found that in patients with cholangitis, taking folic acid and not taking folic acid also showed significant differences in total and direct bilirubin concentrations, and the total and direct bilirubin concentrations in folic acid-treated patients were significantly different. The erythromycin concentration was significantly reduced, thus indicating the effect of folic acid on the treatment of cholangitis.
  • cholangitis is an important pathological feature in the occurrence and development of biliary atresia; on the other hand, cholangitis is the most common complication after biliary atresia surgery; It is used to prevent the occurrence of cholangitis, a complication of biliary atresia, and can also be used in the treatment of cholangitis.
  • Fig. 10, A an additional scRNA transcriptome dataset (Fig. 10, A) was generated using the BD Rhapsody platform, and the analysis was focused on intestinal epithelial cells (Fig. 10, B).
  • FOLH1 folate uptake
  • FTH1, NCOA4, SLC40A1 iron export
  • NFE2L2/NRF2, SELENOP, GPX4 inhibition of lipid peroxidation
  • STAT1, IFITM2, IFITM3 were significantly reduced in patients receiving folic acid treatment (C in FIG. 10 , A, B in FIG. 11 ).
  • Folic acid treatment also significantly increased the concentration of tetrahydrobiopterin (BH4) in the circulation, but decreased the concentration of homocysteine in the liver (Fig. 11, C).
  • Increased expression of SLC40A1 in intestinal epithelial cells demonstrates correction of systemic iron overload and luminal iron deficiency.
  • plasma iron levels decreased, but fecal iron levels increased in 10 cases (Fig. 12A).
  • Other therapeutic benefits of folic acid treatment include suppression of eosinophilic infiltration in the small intestine, suppression of IgG-Ro/SSA autoantibody production, and increased IgM/IgG4 ratio (Fig. 12, B), which may predict liver health and small bowel and liver marrow in BA Inhibition of the IFN-I signaling pathway in cells.
  • folic acid supplementation can reduce the total and direct bilirubin concentrations in patients with biliary atresia, achieving the effect of preventing and/or treating cholangitis.
  • folic acid supplementation can also correct systemic iron overload and luminal iron deficiency, inhibit eosinophil infiltration in the small intestine, inhibit the production of IgG-Ro/SSA autoantibodies, increase the ratio of IgM/IgG4, and at the same time, prolong the period of autoimmunity and improve liver function.
  • mice After the mice were anesthetized, they were killed by dislocation. The mice were placed in a beaker filled with 75% ethanol and soaked for 5 minutes, and the soaked animals were absorbed with paper to absorb excess alcohol. Tear the skin directly to the mouse calf joint by hand, remove the mouse foot joint and skin; use scissors to remove the hind limb along the greater trochanter of the mouse thigh, remove the muscle tissue and place it in a petri dish containing 75% ethanol After soaking for 5 minutes, replace with a new 75% ethanol Petri dish and move it into an ultra-clean bench.
  • liquid A Dilute poly(I:C) with 200 ⁇ l Opti-MEM (poly(I:C) (polyinosinic acid cytidylic acid) is a synthetic double-stranded RNA (dsRNA) analog that mimics double-stranded RNA virus infection (stimulatory Concentration 5ug/ml)) or poly dAdT (Poly(dA:dT) (polydeoxyadenylic acid), is a double-stranded alternating copolymer DNA model, simulating DNA virus infection (5ug/ml));
  • B liquid use Dilute 10 ⁇ l lipo2000 with 200 ⁇ l Opti-MEM, mix A solution and B solution gently, let stand for 5min, draw B solution into A solution, mix gently, stand at room temperature for 20min
  • Bacteria, antisense RNA viruses, DNA viruses, etc. can cause excessive activation of the I-IFN pathway in vivo, and the above bacteria and/or viruses can be inhibited by supplementing folic acid or its derivatives (leucovorin calcium)
  • the excessive activation of I-IFN caused by infection can prevent and/or treat bacterial and/or viral infection by supplementing folic acid or its derivatives (leucovorin calcium), so as to achieve the effect of preventing and/or treating neonatal infectious diseases.
  • folic acid or its derivatives combined with at least one of the active ingredients vitamin B, vitamin C and taurine, especially combined with vitamin B6, vitamin B12, etc., exhibited More excellent inhibitory effect against the above-mentioned bacterial and viral infections.
  • mice (3) Observe the survival status of the mice: the incidence of jaundice and the growth weight of the mice.
  • RRV Infect rhesus monkey rotavirus
  • the jaundice rate of the control mouse group was 0%
  • the jaundice rate of the RRV-induced BA mouse model group was 100%
  • the jaundice rate of the folic acid and vitamin B6 supplemented group was 45%
  • the RRV The jaundice rate of the induced BA mouse model group was significantly different from that of the folic acid and vitamin B6 supplemented group (p ⁇ 0.05). It can be seen that the BA model mouse model was successfully established, while the folic acid and vitamin B6 supplemented group significantly reduced The rate of jaundice in BA model mice was reduced from 100% in the RRV group to 45%, showing a significant therapeutic effect.
  • Example 9 Folic acid reduces the occurrence of food allergy in patients with biliary atresia and choledochal cyst
  • the survey was conducted by Guangzhou Women and Children's Medical Center (GWCMC) Pediatric Surgery Joint Institute of Pediatrics, using electronic forms to obtain information.
  • the sample contained a total of 249 valid cases, of which 154 were from BA patients and 95 were from CC patients. Participants are recruited through an online mini-program (compatible with computers and mobile phones). Participants were invited to complete an online survey.
  • the main content of the structured questionnaire included mother's nutritional intake during pregnancy and postpartum, postpartum nutrition and infection, postoperative complications and infant allergens.
  • the survey has been approved by the GWCMC Medical Ethics Committee for data collection and analysis.
  • folic acid therapy was used for 7 days before surgery, and intestinal specimens were taken for immunofluorescence staining. It was also found that the number of eosinophils involved in food allergy in the intestine was significantly reduced, which indicated that, Folic acid also has preventive and inhibitory effects on the occurrence of food allergy in patients with choledochal cyst.
  • Example 10 Folic acid is used to suppress necrotizing enterocolitis
  • mice of SPF grade were gavaged with mouse milk replacer, and then placed in 90% N 2 +5% CO 2 +5% O 2 , 10min at 4°C, then take it out.
  • the NEC model mice were divided into groups: (1) normal mouse group, normal breast-fed mice; (2) NEC model mouse group; (3) folic acid+NCE model mouse group, given on the basis of NEC model mice And folic acid, wherein, the folic acid dosage is 6mg/kg, intraperitoneal injection, 16 hours of administration, administration once a day, the experiment is terminated on the 6th day. After the termination, the mice were sacrificed, and the intestinal tissue was collected for observation, and the RNA of the intestinal tissue was extracted, and qPCR was performed to detect the expression levels of IFN- ⁇ and IL-1 ⁇ in the intestinal tissue.
  • folic acid has obvious preventive and therapeutic effects on NEC.
  • Example 11 Folic acid is used for the prevention and treatment of neonatal cytomegalovirus infection (HCMV)
  • the detection index is the copy number of HCMV in the urine.
  • the copy number of urinary HCMV in the group taking folic acid was controlled in a shorter period of time, the average days were shortened by 1-2 days, and the blood of the children was collected on days 3, 6, and 10 for detection of inflammatory factors , found that the levels of TNF- ⁇ and IL-1 ⁇ inflammatory factors in the folic acid group were lower, especially on the 6th day, and the difference was significant.
  • Neonatal cytomegalovirus infection affects the development of infants and may cause damage to the liver, central nervous system, etc., especially, therefore, the present invention demonstrates that folic acid can inhibit cytomegalovirus infection by proving that folic acid is used for neonatal Prospects for the prevention and treatment of cytomegalovirus infection.
  • the present invention proves that serum folic acid significantly reduces in BA (A in Fig. 1), and the level of folic acid can effectively distinguish BA from non-BA subjects (B in Fig. 1), so folic acid supplementation during pregnancy can effectively To prevent biliary atresia; folic acid supplementation can increase BA body weight (Figure 2, B), can reduce the rate of jaundice (Figure 2, C), at the same time can anti-inflammatory, reduce liver tissue damage and fibrosis (Figure 2, D) , reduce the level of IFN- ⁇ (E in Figure 2), repair intestinal damage ( Figure 3, 4); the expression of intestinal tissue inflammatory factors (S100a8, S100a9, IFN- ⁇ ) decreased while the expression of anti-inflammatory gene (Nox2) increased ( Figure 5), S100a8, S100a9, IFN- ⁇ or Nox2 levels can effectively distinguish BA from non-BA subjects; and, the present invention also verifies that at least two of S100a8, S100a9, IFN- ⁇ , Nox2, folic acid The
  • folic acid or its salt can effectively prevent and treat biliary atresia, cholangitis, and pathological jaundice
  • the present invention proves that supplementing folic acid can reduce the jaundice rate in the virus-infected biliary atresia mouse model, improve mouse liver/intestinal function, Inhibiting the expression of hepatic cytotoxic cytokine IFN- ⁇ can also reduce the total and direct bilirubin concentrations in patients with biliary atresia (B, C in Figure 9), achieving the effect of preventing and/or treating cholangitis ( C in Figure 8, A in Figure 9).
  • folic acid supplementation can also correct systemic iron overload and luminal iron deficiency, inhibit the infiltration of eosinophils in the small intestine, inhibit the production of IgG-Ro/SSA autoantibodies, and increase the ratio of IgM/IgG4 ( Figure 12, B).
  • Prolong autoimmune phase and liver survival rate The present invention also proves: Supplement calcium folinate (CF) can reduce the content of INF ⁇ , ROS in the liver (D in Fig. 7), reduces the incidence of jaundice and improves neonatal growth (Fig.
  • the present invention also provides the use of one or more of folic acid, S100a8, S100a9, Nox2, and IFN- ⁇ as a diagnostic or auxiliary diagnostic marker for biliary atresia. Therefore, the present invention not only finds that folic acid can be used for diagnosing, preventing and treating diseases such as biliary atresia, but also expands its application prospect by exploring the mechanism.

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Abstract

本发明涉及生物医药技术领域，公开了叶酸在预防、诊断和治疗遗传性、感染性或过敏性疾病中的应用。本发明发现叶酸通过改善炎症、调节铁离子代谢、纠正肠道菌群紊乱、减轻肝脏/肠道组织损伤、抑制炎症因子的表达，促进Nox2的表达，达到对遗传性、感染性或过敏性疾病的防治作用。同时，本发明提供了叶酸、S100a8、S100a9、Nox2、IFN-γ中的一种或多种作为胆道闭锁的诊断或辅助诊断标志物的应用。同时，提供了叶酸或其衍生物，制备为食品、营养制剂或药物，应用于儿童或成人，达到防治胆道闭锁、胆管炎、黄疸、感染性疾病、肠道疾病等相关疾病以及叶酸代谢异常所导致的疾病的目的。

Description

叶酸在预防、诊断和治疗遗传性、感染性或过敏性疾病中的应用 技术领域
本发明属于生物医药技术领域，具体涉及叶酸在预防、诊断和治疗遗传性、感染性或过敏性疾病中的应用。
背景技术
胆道闭锁(biliary atresia，以下有时简称为“BA”)是引起婴幼儿阻塞性黄疸的常见疾病。婴幼儿围生期(例如，孕28周至出生后4周)由于病毒感染(已知的与BA发生有关的病毒包括人疱疹病毒、巨细胞病毒、轮状病毒、呼肠孤病毒等)等因素激发引起胆管上皮细胞凋亡或坏死、胆管受损、炎症和纤维化等病理变化的自身免疫反应，预后差，病死率高，病因及发病机理至今未明。胆道闭锁的基本病理变化例如为肝内、外胆管进行性炎症和肝纤维化，其肝纤维化的发展比其他成人疾患更快且更具有侵袭性，虽然肝外胆道梗阻通过Kasai手术能部分缓解症状，延缓病情进展，但大多数患儿仍因术后肝内胆管炎症呈进行性发展，最终导致肝硬化和门脉高压，甚至肝衰竭，成为危及患儿生命的严重疾病。目前胆道闭锁是儿童时期肝移植的首要原因。此外，胆道闭锁疾病与其他出生黄疸持续不退的婴幼儿难以区分，在临床中需要通过肝穿刺等有创方式进行确诊，且除了进行葛西手术、肝移植等手术方式也尚无有效的药物可医治，在我国，70％以上患儿往往由于确诊时间较长延误了手术最佳治疗窗而最终不幸身亡。
新生儿感染性疾病，也称为新生儿感染，是一种常见的新生儿疾病，也是导致新生儿死亡的一个重要因素，其中包括了肺部感染、脐带感染、脑部感染、皮肤感染、泌尿系统感染、口腔感染等，感染途径包括了母体宫内感染、产时感染、出生后感染等。
叶酸，又称维生素M、维生素Bc、维生素B9，是一种水溶性维生素，一般正常人叶酸的需求量为200～400μg/d，世界卫生组织建议成人至少为200μg/d，孕妇和母乳为400μg/d，一般超出成人最低需要量20倍也不会引起中毒。叶酸作为肠道乳酸双歧杆菌的代谢产物，在体内发挥重要的生理作用，包括维持红细胞的生成以及功能等，而叶酸对胆道闭锁疾病的研究未见报道。
发明内容
为了解决上述问题，本发明对遗传性、感染性或过敏性疾病的发生机制、疾病伴随的肠道功能紊乱、肝纤维化等并发症、相关疾病胆道闭锁、胆管炎、黄疸、病毒性感染等的诊断、预防和治疗进行了深入研究，得到了如下结果：
本发明发现，叶酸在BA患者的血清中显著降低，可以作为BA的诊断或辅助诊断的标志物。在病毒感染性胆道闭锁小鼠模型中使用叶酸，可以显著增加小鼠体重、降低小鼠的黄疸率、改善小鼠肝脏功能、抑制肠道或肝脏中炎症细胞因子S100a8、S100a9和IFN-γ的表达，促进髓源性抑制细胞特征性转录因子Nox2的表达；同时，叶酸还能显著地改善肠道炎症、调节铁离子代谢、纠正肠道菌群紊乱、减轻肝脏、肠道组织损伤。因此，叶酸可通过改善抗炎、调节铁离子代谢、纠正肠道菌群紊乱、改善肝损伤等机理，达到对胆道闭锁、胆管炎、肝纤维化、胃肠道菌群紊乱、炎症性肠病、病毒感染的诊断和防治作用。叶酸还可以降低胆道闭锁患者的总胆红素浓度和直接胆红素浓度，达到预防和/或治疗胆管炎的效果。同时，补充叶酸还可以达到纠正全身铁超载和管腔铁缺乏、抑制小肠嗜酸性粒细胞浸润、抑制IgG-Ro/SSA自身抗体的产生，增加IgM/IgG4比值，同时，延长自身免疫期和改善肝脏功能；本发明还证明了：补充亚叶酸钙(CF)可降低肝脏中的INFβ、ROS的含量，降低黄疸发生率并改善新生儿生长；提高小肠中Dhfr、Mthfr、Nrf2、Slc40a1的表达量，从而使叶酸代谢、氧化应激和铁稳态得到恢复；降低肝脏中CD4 +T细胞、CD8 +T细胞中的IFN-γ含量、浆细胞中 IgG1含量；降低肝脏门管区周围淋巴细胞浸润；增加肝小叶结构的完整性；使肠上皮细胞的气球样变性得到恢复，达到治疗胆道闭锁的效果；通过补充叶酸或其衍生物(亚叶酸钙)可以预防和/或治疗细菌和/或病毒感染，从而达到预防和/或治疗新生儿感染疾病的效果。此外，根据S100a8、S100a9、IFN-γ、Nox2以及叶酸在BA和非BA中的表达情况，本发明首次公开了叶酸、S100a8、S100a9、IFN-γ、Nox2中的至少一种作为胆道闭锁的诊断或辅助诊断的标志物的用途。
本发明还公开了叶酸或其衍生物单独地与其他维生素B联合用于胆道闭锁、胆管炎、黄疸、食物过敏、细菌感染、病毒感染、坏死性小肠结肠炎的预防和治疗，并且对于胆道闭锁导致的黄疸、肝脏炎症性疾病、肝脏功能损伤、肠道修复等方面均表现出了显著效果。
本发明第一个方面的目的，在于提供叶酸或其衍生物在制备药物中的应用。
本发明的第二方面的目的，在于提供叶酸或其衍生物在制备体外非治疗目的产品中的应用。
本发明的第三方面的目的，在于提供诊断或辅助诊断胆道闭锁疾病的标志物。
本发明的第四方面的目的，在于提供检测本发明第三方面的标志物的试剂在制备诊断或辅助诊断胆道闭锁的产品中的应用。
本发明的第五方面的目的，在于提供一种用于诊断或辅助诊断胆道闭锁疾病的试剂盒。
本发明的第六方面的目的，在于提供叶酸或其衍生物在制备预防和/或治疗病毒性感染的药物中的应用。
本发明的第六方面的目的，在于提供叶酸或其衍生物在制备预防和/或治疗新生儿感染的药物中的应用。
本发明的第七方面的目的，在于提供叶酸或其衍生物在制备预防和/或治疗肝肠循环障碍所导致的疾病的药物中的应用。
本发明的第八方面的目的，在于提供叶酸或其衍生物在制备预防和/或治疗炎症性肠病的药物中的应用。
本发明的第九方面的目的，在于提供叶酸或其衍生物在制备调节肠道菌群的产品中的应用。
本发明的第十方面的目的，在于提供叶酸或其衍生物在制备预防和/或治疗S100a8或S100a9表达升高导致的疾病的药物中的应用。
本发明的第十一方面的目的，在于提供叶酸或其衍生物在制备预防和/或治疗缺铁性贫血的产品中的应用。
本发明的第十二方面的目的，在于提供叶酸或其衍生物在制备预防和/或治疗IFN-γ表达升高导致的疾病的药物中的应用。
本发明的第十三方面的目的，在于提供一种含有叶酸或其衍生物的产品的制备方法。
本发明的第十四方面的目的，在于提供一种药物。
本发明的第十五方面的目的，在于提供一种诊断方法。
本发明的第十六方面的目的，在于提供一种预防和/或治疗疾病的方法。
为了实现上述技术方案，本发明的第一个方面，提供叶酸或其衍生物在制备药物中的应用；
所述药物具有(a1)～(a9)中至少一种功能：
(a1)防治胆道闭锁；
(a2)防治胆管炎；
(a3)防治新生儿感染性疾病；
(a4)改善全身铁超载；
(a5)抑制小肠嗜酸性粒细胞浸润；
(a6)抑制IgG-Ro/SSA自身抗体的表达；
(a7)IgM/IgG4比值；
(a8)延长自身免疫期；
(a9)提高肝功能。
本发明的第二个方面，提供叶酸或其衍生物在(b1)～(b5)中任一种中的应用；
(b1)制备体外非治疗目的地抑制炎症因子表达的产品；
(b2)制备体外非治疗目的地促进髓源性抑制细胞特征性转录因子表达的产品；
(b3)制备体外非治疗目的地促进铁离子转运蛋白SLC11A2表达的产品；
(b4)制备体外非治疗目的地抑制铁离子转运蛋白SLC40A1表达的产品；
(b5)制备体外非治疗目的地促进叶酸转运蛋白表达的产品。
优选地，所述炎症因子包含S100a8、S100a9和IFN-γ中的至少一种。
优选地，所述髓源性抑制细胞特征性转录因子包含Nox2。
优选地，所述叶酸转运蛋白包含SLC46A1。
本发明的第三个方面，提供诊断或辅助诊断胆道闭锁疾病的标志物，包含叶酸、S100a8、S100a9、Nox2、IFN-γ中的至少一种。
优选地，所述标志物包含：
a)叶酸；以及
b)S100a8、S100a9、Nox2、IFN-γ中的至少一种。
优选地，所述S100a8、S100a9、Nox2和IFN-γ包括蛋白质和/或mRNA。
优选地，所述标志物来自体液、血液、组织、细胞或排泄物；进一步优选地，所述标志物来自血液、组织或排泄物。
优选地，所述组织包括肝脏、肠道、胚胎肝脏、胚胎肠道。
优选地，所述排泄物包括粪便、尿液。
优选地，所述血液为全血、血清或血浆；进一步为血清。
优选地，所述叶酸来自血液。
优选地，所述S100a8、S100a9、Nox2和IFN-γ来自组织；进一步优选地，所述S100a8、S100a9、Nox2和IFN-γ来自肝脏组织或肠道组织；更进一步优选地，所述S100a8、S100a9、Nox2、IFN-γ来自肠道组织。
优选地，当待测对象满足(c1)～(c5)中至少一项，则诊断为胆道闭锁疾病患者；
(c1)叶酸相对于参考水平显著降低；
(c2)S100a8相对于参考水平显著升高；
(c3)S100a9相对于参考水平显著升高；
(c4)IFN-γ相对于参考水平显著升高；
(c5)Nox2相对于参考水平显著降低；
所述参考水平为未患有胆道闭锁疾病的同年龄段受试者的水平；所述同年龄段受试者为健康人或胆总管囊肿患者。
本发明的第四个方面，提供检测本发明第三方面的标志物的试剂在制备诊断或辅助诊断胆道闭锁的产品中的应用。
优选地，所述产品包括试剂、试剂盒、基因芯片、蛋白质芯片。
优选地，所述产品用于执行以下检测方法中的至少一种：
(d1)比色法；(d2)化学发光法；(d3)原子吸收分光光度法；(d4)聚合酶链反应；(d5)微数字聚合酶链反应；(d6)荧光聚合酶链反应；(d7)环介导等温扩增反应；(d8)酶联免疫吸附测定法；(d9)核苷酸序列测序法；(d10)氨基酸序列测序法；(d11)变性梯度凝胶电泳；(d12)核酸分型芯片检测；(d13)高效液相色谱法；(d14)原位杂交；(d15)生物质谱法；(d16)高分辨率溶解曲线分析；(d17)单链构象异构多态分析；(d18)探针扩增阻滞突变系统分析。
优选地，当检测标志物为S100a8、S100a9、Nox2和IFN-γ的mRNA时，所述产品含有检测所述标志物和/或内参mRNA的引物、和/或检测探针。
进一步优选地，当检测标志物为S100a8、S100a9、Nox2和IFN-γ的mRNA时，所述产品还包含：样品的处理试剂，包括但不限于样品裂解试剂、样品纯化试剂和/或样品核酸提取试剂。
更进一步优选地，当检测标志物为S100a8、S100a9、Nox2和IFN-γ的mRNA时，所述产品还包含：RNA提取试剂、dNTP、DNA聚合酶、双链特异性荧光染料以及水中的一种或多种。
优选地，当检测标志物为S100a8mRNA，所述产品包括检测S100a8mRNA的引物，所述引物的核苷酸序列如SEQ ID NO.1和SEQ ID NO.2所示。
优选地，当检测标志物为S100a9mRNA，所述产品包括检测S100a9mRNA的引物，所述引物的核苷酸序列如SEQ ID NO.3和SEQ ID NO.4所示。
优选地，当检测标志物为IFN-γmRNA，所述产品包括检测IFN-γmRNA的引物，所述引物的核苷酸序列如SEQ ID NO.5和SEQ ID NO.6所示。
优选地，当检测标志物为Nox2 mRNA，所述产品包括检测Nox2 mRNA的引物，所述引物的核苷酸序列如SEQ ID NO.7和SEQ ID NO.8所示。
优选地，所述产品的检测样本或受试样本来自待测对象的来自体液、血液、组织、细胞或排泄物；进一步优选地，所述标志物来自血液、组织或排泄物。
优选地，所述组织包括肝脏、肠道、胚胎肝脏、胚胎肠道。
优选地，所述排泄物包括粪便、尿液。
优选地，所述血液为全血、血清或血浆、干血斑；进一步为血清、干血斑。
优选地，所述待测对象为成人或儿童。
优选地，所述儿童包括出生28天内的新生儿、1岁以内的婴儿、1～6岁的幼儿、以及6～8岁的儿童。
优选地，所述成人选自妊娠期的女性成年人、围产期的女性成年人或哺乳期的女性成年人。
优选地，当待测对象满足(c1)～(c5)中至少一项，则诊断为胆道闭锁疾病患者；
(c1)叶酸相对于参考水平显著降低；
(c2)S100a8相对于参考水平显著升高；
(c3)S100a9相对于参考水平显著升高；
(c4)IFN-γ相对于参考水平显著升高；
(c5)Nox2相对于参考水平显著降低。
所述参考水平为未患有胆道闭锁疾病的同年龄段受试者的水平；所述同年龄段受试者为健康人或胆总管囊肿患者。
本发明的第五方面，在于提供一种用于诊断或辅助诊断胆道闭锁疾病的试剂盒，包含检测检测本发明第三方面的标志物的试剂。
优选地，当检测标志物为S100a8、S100a9、Nox2和IFN-γ的mRNA时，所述产品含有检测所述标志物和/或内参mRNA的引物、和/或检测探针。
进一步优选地，当检测标志物为S100a8、S100a9、Nox2和IFN-γ的mRNA时，所述产品还包含：样品的处理试剂，包括但不限于样品裂解试剂、样品纯化试剂和/或样品核酸提取试剂。
更进一步优选地，当检测标志物为S100a8、S100a9、Nox2和IFN-γ的mRNA时，所述产品还包含：RNA提取试剂、dNTP、DNA聚合酶、双链特异性荧光染料以及水中的一种或多种。
优选地，当检测标志物为S100a8mRNA，所述产品包括检测S100a8mRNA的引物，所述引物的核苷酸序列如SEQ ID NO.1和SEQ ID NO.2所示。
优选地，当检测标志物为S100a9mRNA，所述产品包括检测S100a9mRNA的引物，所述引物的核苷酸序列如SEQ ID NO.3和SEQ ID NO.4所示。
优选地，当检测标志物为IFN-γmRNA，所述产品包括检测IFN-γmRNA的引物，所述引物的核苷酸序列如SEQ ID NO.5和SEQ ID NO.6所示。
优选地，当检测标志物为Nox2 mRNA，所述产品包括检测Nox2 mRNA的引物，所述引物的核苷酸序列如SEQ ID NO.7和SEQ ID NO.8所示。
优选地，所述产品的检测样本或受试样本来自待测对象的来自体液、血液、组织、细胞或排泄物；进一步优选地，所述标志物来自血液、组织或排泄物。
优选地，所述组织包括肝脏、肠道、胚胎肝脏、胚胎肠道。
优选地，所述排泄物包括粪便、尿液。
优选地，所述血液为全血、血清或血浆、干血斑；进一步为血清、干血斑。
优选地，所述待测对象为成人或儿童。
优选地，所述儿童包括出生28天内的新生儿、1岁以内的婴儿、1～6岁的幼儿、以及6～8岁的儿童。
优选地，所述成人选自妊娠期的女性成年人、围产期的女性成年人或哺乳期的女性成年人。
优选地，当待测对象满足(c1)～(c5)中至少一项，则诊断为胆道闭锁疾病患者；
(c1)叶酸相对于参考水平显著降低；
(c2)S100a8相对于参考水平显著升高；
(c3)S100a9相对于参考水平显著升高；
(c4)IFN-γ相对于参考水平显著升高；
(c5)Nox2相对于参考水平显著降低；
所述参考水平为未患有胆道闭锁疾病的同年龄段受试者的水平；所述同年龄段受试者为健康人或胆总管囊肿患者。
本发明的第六个方面，提供叶酸或其衍生物在制备预防和/或治疗病毒性感染的药物中的应用。
本发明的第七个方面，提供叶酸或其衍生物在制备预防和/或治疗肝肠循环障碍所导致的疾病的药物中的应用。
本发明的第八个方面，提供叶酸或其衍生物在制备预防和/或治疗炎症性肠病的药物中的应用。
本发明的第九个方面，提供叶酸或其衍生物在制备调节肠道菌群的产品中的应用。
优选地，所述产品包括食品、营养制剂和药物。
优选地，所述产品用于预防、治疗或辅助治疗白色念珠菌性肠炎、葡萄球菌性肠炎、产气荚膜杆菌性急性坏死性肠炎、(新生儿)坏死性小肠结肠炎、败血病、绿脓杆菌肠道感染、变形杆菌肠道感染、肺炎杆菌肠道感染等肠道炎症性疾病或感染性疾病；以及降低由上述疾病导致的早产儿出生风险。
本发明的第十个方面，提供叶酸或其衍生物在制备预防和/或治疗S100a8和/或S100a9表达升高导致的疾病的药物中的应用。
本发明的第十一个方面，提供叶酸或其衍生物在制备预防和/或治疗缺铁性贫血的产品中的应用。
优选地，所述产品包括食品、营养制剂和药物。
本发明的第十二个方面，提供叶酸或其衍生物在制备IFN-γ表达升高导致的疾病的药物中的应用。
本发明的第十三个方面，提供一种含有叶酸或其衍生物的产品的制备方法，将叶酸或其衍生物按比例与辅料混合，得到。
优选地，所述制备方法还包括将得到的混合物根据需求制备成相应形态或剂型。
优选地，所述产品包括食品、营养制剂和药物。
本发明的第十四个方面，提供一种药物，包含：叶酸或其衍生物；
和其他活性成分，所述其他活性成分包含维生素B、维生素C、烟酰胺、氧化锌、维生素A、维生素D、维生素E、维生素K、同型半胱氨酸、谷胱甘肽和牛磺酸中的至少一种。
优选地，所述其他活性成分包含维生素B6、维生素B12中的至少一种。
优选地，所述药物包括：叶酸或其衍生物；和维生素B6、维生素B12、维生素A、维生素D、维生素E、维生素K、维生素C、烟酰胺、氧化锌、牛磺酸中的至少一种。
优选地，所述药物包括叶酸或其衍生物和烟酰胺。
优选地，所述药物包括叶酸或其衍生物和维生素E。
优选地，所述药物包括叶酸或其衍生物和维生素A以及维生素E。优选地，所述药物具有(a1)～(a9)中至少一种功能：
(a1)防治胆道闭锁；
(a2)防治胆管炎；
(a3)防治新生儿感染性疾病；
(a4)改善全身铁超载；
(a5)抑制小肠嗜酸性粒细胞浸润；
(a6)抑制IgG-Ro/SSA自身抗体的表达；
(a7)IgM/IgG4比值；
(a8)延长自身免疫期；
(a9)改善肝功能。
本发明的第十四个方面，提供一种诊断或辅助诊断胆道闭锁疾病的方法，包括如下步骤中的至少一种：
(e1)检测本发明第三个方面的标志物的水平和/或活性；
(e2)采用本发明第五个方面的试剂盒。
本发明的第十五个方面，提供一种方法，包括如下步骤中的至少一种：
(f1)向受试者施用叶酸或其衍生物；
(f2)向受试者施用本发明第十三个方面的药物。
所述方法用于(a1)～(a16)中至少一种：
(a1)防治胆道闭锁；
(a2)防治胆管炎；
(a3)防治新生儿感染性疾病；
(a4)改善全身铁超载；
(a5)抑制小肠嗜酸性粒细胞浸润；
(a6)抑制IgG-Ro/SSA自身抗体的表达；
(a7)IgM/IgG4比值；
(a8)延长自身免疫期；
(a9)提高肝功能；
(a10)防治病毒性感染；
(a11)防治肝肠循环障碍所导致的疾病；
(a12)防治炎症性肠病；
(a13)调节肠道菌群；
(a14)防治S100a8或S100a9表达升高导致的疾病；
(a15)防治缺铁性贫血；
(a16)防治IFN-γ表达升高导致的疾病。
优选地，所述方法包括如下步骤中的至少一种：
(g1)向受试者施用有效量的叶酸或其衍生物；
(g2)向受试者施用有效量的本发明第十三个方面的药物。
优选地，所述方法用于(a1)～(a9)中至少一种：
(a1)防治胆道闭锁；
(a2)防治胆管炎；
(a3)防治新生儿感染性疾病；
(a4)改善全身铁超载；
(a5)抑制小肠嗜酸性粒细胞浸润；
(a6)抑制IgG-Ro/SSA自身抗体的表达；
(a7)IgM/IgG4比值；
(a8)延长自身免疫期；
(a9)提高肝功能。
根据本发明的上述方面：
优选地，叶酸的分子式为C 19H 19N 7O 6，分子量为441.4，CAS为59-30-3。
优选地，所述衍生物包括叶酸在药学上可以接受的盐、酯、水合物、溶剂化物、多晶型物、互变异构体、前药和叶酸功能等价物；进一步优选地，所述衍生物包括(a1)～(a2)中任一种：(a1)亚叶酸、二氢叶酸、四氢叶酸、5-甲基四氢叶酸、5,10-亚甲基四氢叶酸、5,10-亚甲基四氢叶酸、5,10-甲酰亚氨基四氢叶酸、5-甲酰四氢叶酸、10-甲酰四氢叶酸和10-甲基四氢叶酸；(a2)(a1)上药学上可接受的盐，包括但不限于钙、钠、锌、精氨酸、胆碱、乙酰胆碱、N-甲基氨基乙醇、2-氨基-2-甲基-丙醇、1,1-二甲基双胍、苯乙基双胍、二氨基胍、葡糖胺和二甲基氨基乙醇。
优选地，所述衍生物包含叶酸在药学上可接受的盐、亚叶酸、亚叶酸在药学上可接受的盐中的至少一种。
优选地，所述胆管炎包含细菌性胆管炎、病毒性胆管炎。
优选地，所述新生儿感染包含：细菌感染、病毒感染中的至少一种。
优选地，所述病毒包含：DNA病毒和RNA病毒中的至少一种。
优选地，所述RNA病毒包含：反义RNA病毒和双链RNA病毒中的至少一种。
优选地，所述新生儿感染性疾病包含：I型干扰素通路的过度激活引起的疾病。
优选地，所述新生儿感染性疾病包括：新生儿肝炎综合征、新生儿败血症、新生儿肺炎、新生儿感染导致的胃肠道疾病。
优选的，所述细菌感染性疾病包括大肠杆菌、金黄色葡萄球菌或溶血性链球菌。
优选的，所述病毒感染性疾病包括巨细胞病毒(CMV)感染、轮状病毒感染、疱疹病毒感染；其中，所述病毒性感染疾病还包括病毒性腹泻。
所述药物的剂型为儿童适用的剂型或成人适用的剂型。
优选地，所述儿童包括出生28天内的新生儿、1岁以内的婴儿、1～6岁的幼儿、以及6～18岁的儿童。
优选地，所述成人包括妊娠期的女性成年人、围产期的女性成年人和哺乳期的女性成年人。
优选地，所述剂型包括胶囊剂、片剂、微囊制剂、注射剂、栓剂、喷雾剂、散剂、软胶囊剂、滴丸、蜜丸、丸剂、颗粒剂、蜜炼膏剂、缓控释制剂、口服液体制剂、注射剂、咀嚼片、口腔片、透皮贴剂和泡腾片。
优选地，所述叶酸或其衍生物在所述药物中的单位剂量为0.1mg～1g。
优选地，所述叶酸或其衍生物在所述药物中的单位剂量为0.1mg、0.2mg、0.3mg、0.4mg、0.5mg、0.6mg、0.7mg、0.8mg、0.9mg或1.0mg。
优选地，所述叶酸或其衍生物在所述药物中的单位剂量为0.2mg、0.4mg、0.8mg、或1.0mg。
优选地，所述药物还包含其他活性成分，所述其他活性成分为维生素B、维生素C、烟酰胺、氧化锌、维生素A、维生素E、同型半胱氨酸、谷胱甘肽和牛磺酸中的至少一种，其中，维生素B保肝护肝；维生素C，又称为L-抗坏血酸，它是一种抗氧化剂，同时也是一种辅酶；烟酰胺，又称尼克酰胺，是烟酸的酰胺化合物；氧化锌是一种锌的氧化物；维生素A是一种脂溶性维生素；维生素E又名生育酚或产妊酚，是一种主要的抗氧化剂之一；同型半胱氨酸，又称高半胱氨酸，是一种含硫氨基酸；谷胱甘肽(GSH)是一种含γ-酰胺键和巯基的三肽，由谷氨酸、半胱氨酸及甘氨酸组成，具有抗氧化、解毒作用；牛磺酸保肝护肝，促进胃肠功能、增加人体免疫力、提高机体抵抗力、抗病能力；它们与叶酸组合使用达到促进叶酸吸收、协同抗病等目的。
优选地，所述维生素B为维生素B1(硫铵素)、维生素B2(核黄素)、维生素B3(烟酸)、维生素B5(泛酸)、维生素B6(吡哆醇)、维生素B7(生物素)和维生素B12(钴胺素)中的至少一种。
优选的，在一个实施方式中，所述药物包括：叶酸或其衍生物；和维生素B6、维生素B12、维生素A、维生素E、维生素C、烟酰胺、氧化锌、牛磺酸中的至少一种。
优选地，所述药物还包含药物辅料，所述药物辅料包括稀释剂、填充剂、赋形剂、赋形剂、粘合剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、吸附载体、润滑剂和香味剂中的一种或多种。
优选地，所述载体或赋形剂可选自乳糖水合物、微晶纤维素、甘露醇、柠檬酸钠、磷酸钙、甘氨酸和淀粉中的一种或多种；崩解剂可选自交联聚维酮、共聚维酮、羟基乙酸淀粉钠、交联羧甲基纤维素钠和特定的复合硅酸盐中的一种或多种；粘合剂可选自聚乙烯吡咯烷酮、羟丙基甲基纤维素(HPMC)、羟丙基纤维素(HPC)、蔗糖、明胶和阿拉伯胶中的一种或多种。
优选地，所述病毒性感染中的病毒包括但不限于人疱疹病毒、巨细胞病毒、轮状病毒和呼肠孤病毒。
优选地，所述肝肠循环障碍所导致的疾病包括高胆固醇血症、胆固醇结石、黄疸和肝纤维化。
优选地，所述炎症性肠病包括未分化性结肠炎、溃疡性结肠炎和克罗恩病。
优选地，所述S100a8和/或S100a9表达升高导致的疾病包括但不限于系统性红斑狼疮(systemiclupus erythematosus，SLE)、类风湿关节炎(rheumatoid arthritis，RA)、炎性肠病((inflammatorybowel disease，IBD)、原发性肾小球疾病、系统性硬化症(systemic sclerosis，SSc)、多发性硬化(MS,multiple sclerosis,实验性自身免疫性脑脊髓炎、experimental autoimmune encephalomye-litis EAE)、COPD慢性阻塞性肺疾病、骨髓白细胞生成、骨髓增生异常综合征、肿瘤发生、胃癌、乳腺癌、黑色素瘤、牙周炎症性疾病、肥胖、2型糖尿病(type2diabetes mellitus,T2DM)及其并发症、心血管疾病(心肌细胞功能障碍、心肌梗死、动脉粥样硬化)和皮肤病。
优选地，所述缺铁性贫血包含小儿缺铁性贫血。
优选地，所述IFN-γ表达升高导致的疾病包含儿童或成人的自身免疫性疾病。
优选地，所述自身免疫性疾病包括但不限于：I型糖尿病、再生障碍性贫血(AA)、类风湿关节炎(rheumatoid arthritis，RA)、系统性红斑狼疮(systemic lupus erythematosus，SLE)、自身免疫性甲状腺疾病(AITD)(包括graves病(GD)，桥本甲状腺炎(HT,Hashimoto's Thyroiditis))、重症肌无力(MG)、胰岛素依赖性糖尿病(IDDM)、特发性血小板减少性紫癜(ITP)、强直性脊柱炎(Ankylosing spondylitis)、干燥综合征(Sjogren's syndrome,SS)、自身免疫性肝病(AILD)、急性播散性(传播性)脑脊髓炎(Acute disseminated encephalomyelitis，ADE，实验性自身免疫脑脊髓炎MOG)。
优选地，所述食品、营养制剂包括常规配方食品、特殊医学用途的配方食品、肠内营养制剂和肠外营养 制剂。
优选地，常规配方食品包括婴儿配方奶粉、孕期和哺乳期配方奶粉中的至少一种。
优选地，特殊医学用途配方食品包括早产/低出生体重婴儿配方奶粉、无乳糖配方或低乳糖配方奶粉、乳蛋白部分水解配方奶粉、乳蛋白深度水解配方或氨基酸配方奶粉、母乳和婴儿配方食品的强化剂或营养补充剂、适用于食品不耐受性、变态反应、肝病特殊配方食品、疾病和功能障碍的配方食品中的至少一种；特殊医学用途配方食品(Food for Special Medical Purpose，FSMP)，是为了满足进食受限、消化吸收障碍、代谢紊乱或特定疾病状态人群对营养素或膳食的特殊需要，专门加工配制而成的配方食品，该类产品必须在医生或临床营养师指导下，单独食用或与其他食品配合食用。特殊医学用途配方食品属于特殊膳食用食品，当目标人群无法进食普通膳食或无法用日常膳食满足其营养需求时，特殊医学用途配方食品可以作为一种营养补充途径，对治疗、康复及机体功能维持等方面起着重要的营养支持作用。
优选地，肠外营养制剂包括脂肪乳注射液、全合一营养液和静脉注射液中的至少一种。
优选地，肠内营养制剂包括氨基酸型肠内营养制剂、短肽型肠内营养制剂、整蛋白型肠内营养制剂和组件型肠内营养制剂中的至少一种。
需要说明的是，本发明中叶酸或其衍生物的施用形式不重要，因为本发明实施例已证明，该成分腹腔注射即可达到效果，因而将含叶酸或其衍生物添加至适用于婴幼儿及孕产妇的配方奶粉、特殊医学用途配方食品及其他肠内、肠外营养制剂或药物辅料中即可，只要施用有效量均可实现防治胆道闭锁、肝纤维化、胆管炎、胃肠道菌群紊乱、炎症性肠病、病毒感染、新生儿感染疾病等疾病的作用。
按照感染部位分类，所述新生儿感染还包括新生儿肺部感染(例如新生儿肺炎)、新生儿脐带感染、新生儿脑部感染、新生儿皮肤感染、新生儿泌尿系统感染、新生儿口腔感染、新生儿眼睛感染、新生儿血液感染(例如新生儿败血症)、新生儿胆道感染、新生儿胃部感染、新生儿肠道感染(例如新生儿感染导致的胃肠道疾病)、新生儿肝脏感染(例如新生儿肝炎综合征)、或者新生儿全身多部位并发性感染等。
[1]叶酸或其衍生物在制备预防和/或治疗遗传性、感染性或过敏性疾病的药物中的应用，其中，所述遗传性疾病包括胆道闭锁，所述感染性疾病包括胆管炎、黄疸、新生儿感染、坏死性小肠结肠炎、细菌感染性疾病、病毒感染性疾病，所述过敏性疾病包括食物过敏。
[2]根据上述[1]的应用，所述胆道闭锁包括病毒引起的胆道闭锁、或胆汁淤积引起的胆道闭锁。
[3]根据上述[1]的应用，所述病毒引起的胆道闭锁还包括主要由巨细胞病毒引起的胆道闭锁、或主要由轮状病毒引起的胆道闭锁。
[4]根据上述[1]的应用，所述胆道闭锁还表现为胆道闭锁引起的食物过敏、胆道闭锁引起的黄疸、胆道闭锁引起的胆管炎、胆道闭锁引起的肝脏疾病、胆道闭锁引起的肠道性疾病。
[5]根据上述[4]的应用，所述胆道闭锁引起的肝脏疾病包括胆道闭锁引起的肝脏功能损伤、和/或胆道闭锁引起的肝脏炎症性疾病。
[6]根据上述[4]的应用，所述胆道闭锁引起的肠道性疾病包括胆道闭锁引起的肠道性炎症性疾病。
[7]根据上述[1]的应用，所述黄疸包括病理性黄疸或婴儿期黄疸。
[8]根据上述[7]的应用，所述病理性黄疸包括病毒引起的病理性黄疸、或婴儿期病理性黄疸。
[9]根据上述[7]的应用，所述病理性黄疸还包括病毒引起的婴儿期病理性黄疸；或所述病毒引起的病理性黄疸包括巨细胞病毒性黄疸。
[10]根据上述[1]的应用，所述胆管炎包括细菌性胆管炎、病毒性胆管炎、胆道闭锁引起的胆管炎、胆道闭锁术后并发的胆管炎。
[11]根据上述[10]的应用，所述细菌性胆管炎还包括胆道闭锁引起的细菌性胆管炎、胆道闭锁术后并发的细菌性胆管炎。
[12]根据上述[1]的应用，所述新生儿感染性疾病还包括新生儿细菌感染疾病和新生儿病毒感染疾病、新 生儿支原体或者新生儿衣原体感染；或，所述新生儿感染性疾病包括新生儿肝炎综合征、新生儿败血症、新生儿肺炎、新生儿感染导致的胃肠道疾病。
[13]根据上述[12]的应用，所述新生儿病毒感染还包括RNA病毒和DNA病毒。
[14]根据上述[13]的应用，所述RNA病毒还包括反义RNA病毒、DNA病毒。
[15]根据上述[12]的应用，所述新生儿病毒感染还包括新生儿巨细胞病毒(CMV)感染、新生儿轮状病毒感染、新生儿疱疹病毒感染。
[16]根据上述[1]的应用，所述细菌感染性疾病包括大肠杆菌感染、金黄色葡萄球菌感染或溶血性链球菌感染。
[17]根据上述[1]的应用，所述病毒感染性疾病包括巨细胞病毒(CMV)感染、轮状病毒感染、疱疹病毒感染。
[18]根据上述[17]的应用，所述病毒性感染疾病还包括病毒性腹泻。
[19]根据上述[1]的应用，所述食物过敏包括胆道闭锁引起的或者胆道闭锁伴随的食物过敏、胆总管囊肿引起的或胆总管囊肿伴随的食物过敏；或，所述食物过敏还表现为食物过敏导致的皮肤疾病、食物过敏导致的胃肠道疾病、食物过敏导致的呼吸道疾病。
[20]根据上述[19]的应用，所述食物过敏导致的胃肠道疾病包括食物过敏导致的炎症性肠病。
[21]根据上述[1]的应用，所述叶酸衍生物包括叶酸在药学上可以接受的盐、酯、水合物、溶剂化物、多晶型物、互变异构体、前药和叶酸功能等价物。
[22]根据上述[1]的应用，所述叶酸衍生物包括(a1)～(a2)中任一种：
(a1)亚叶酸、二氢叶酸、四氢叶酸、5-甲基四氢叶酸、5,10-亚甲基四氢叶酸、5,10-亚甲基四氢叶酸、5,10-甲酰亚氨基四氢叶酸、5-甲酰四氢叶酸、10-甲酰四氢叶酸和10-甲基四氢叶酸；
(a2)为(a1)的药学上可接受的盐。
[23]根据上述[21]或[22]的应用，所述药学上的盐包括钙、钠、锌、精氨酸、胆碱、乙酰胆碱、N-甲基氨基乙醇、2-氨基-2-甲基-丙醇、1,1-二甲基双胍、苯乙基双胍、二氨基胍、葡糖胺和二甲基氨基乙醇。
[24]根据上述[1]的应用，所述叶酸衍生物包括亚叶酸钙。
[25]根据上述[1]的应用，所述药物还包含其他活性成分，所述其他活性成分包含维生素B、维生素C、烟酰胺、氧化锌、维生素A、维生素E、维生素D、维生素K、同型半胱氨酸、谷胱甘肽和牛磺酸中的至少一种。
[26]根据上述[25]的应用，所述维生素B包含维生素B1、维生素B2、维生素B3、维生素B5、维生素B6、维生素B7和维生素B12中的至少一种。
[27]根据上述[1]的应用，所述药物包括叶酸或其衍生物、及维生素B6和B12中的至少一种；或所述药物包括：叶酸或其衍生物；和维生素B6、维生素B12、维生素A、维生素E、维生素C、烟酰胺、氧化锌、牛磺酸中的至少一种；或，所述药物包括叶酸或其衍生物和烟酰胺；或，所述药物包括叶酸或其衍生物和维生素E。
[28]根据上述[1]的应用，所述药物的剂型为儿童适用的剂型或成人适用的剂型。
[29]根据上述[28]的应用，所述儿童包括出生28天内的新生儿、1岁以内的婴儿、1～6岁的幼儿、以及6～18岁的儿童。
[30]根据上述[28]的应用，所述成人包括妊娠期的女性成年人、围产期的女性成年人和哺乳期的女性成年人。
[31]根据上述[1]的应用，所述药物的剂型包括胃肠道给药的剂型和非胃肠道给药的剂型。
[32]根据上述[1]的应用，所述药物的剂型包括胶囊剂、片剂、微囊制剂、注射剂、栓剂、喷雾剂、散剂、软胶囊剂、滴丸、蜜丸、丸剂、颗粒剂、蜜炼膏剂、缓控释制剂、口服液体制剂、注射剂、咀嚼片、口腔片、 透皮贴剂和泡腾片。
[33]根据上述[1]的应用，所述叶酸或其衍生物在所述药物中的单位剂量为0.1mg～1g。
[34]根据上述[1]的应用，所述叶酸或其衍生物在所述药物中的单位剂量为0.1mg、0.2mg、0.3mg、0.4mg、0.5mg、0.6mg、0.7mg、0.8mg、0.9mg或1.0mg。
[35]根据上述[1]的应用，所述叶酸或其衍生物在所述药物中的单位剂量为0.2mg、0.4mg、0.8mg、或1.0mg。
[36]一种药物，包含：叶酸或其衍生物；和其他活性成分，所述其他活性成分包含维生素B、维生素C、维生素A、维生素E、维生素D、维生素K、烟酰胺、氧化锌、同型半胱氨酸、谷胱甘肽和牛磺酸中的至少一种。
[37]根据上述[36]的药物，所述药物包括：叶酸或其衍生物；和维生素B，所述维生素B包含维生素B1、维生素B2、维生素B3、维生素B5、维生素B6、维生素B7和维生素B12中的至少一种。
[38]根据上述[36]的药物，所述药物包括叶酸或其衍生物、及维生素B6和B12中的至少一种；或，所述药物包括：叶酸或其衍生物；和维生素B6、维生素B12、维生素A、维生素E、维生素C、维生素D、维生素K、烟酰胺、氧化锌、牛磺酸中的至少一种；或，包括叶酸或其衍生物，和烟酰胺；或，包括叶酸或其衍生物，和维生素E；或，包括叶酸或其衍生物，和维生素A和维生素E。
[39]根据上述[36]-[38]的药物，所述叶酸衍生物包括叶酸在药学上可以接受的盐、酯、水合物、溶剂化物、多晶型物、互变异构体、前药和叶酸功能等价物。
[40]根据上述[36]-[38]的药物，所述叶酸衍生物包括(a1)～(a2)中任一种：
(a1)亚叶酸、二氢叶酸、四氢叶酸、5-甲基四氢叶酸、5，10-亚甲基四氢叶酸、5，10-亚甲基四氢叶酸、5，10-甲酰亚氨基四氢叶酸、5-甲酰四氢叶酸、10-甲酰四氢叶酸和10-甲基四氢叶酸；
(a2)为(a1)的药学上可接受的盐。
[41]根据上述[40]的的药物，所述药学上的盐包括钙、钠、锌、精氨酸、胆碱、乙酰胆碱、N甲基氨基乙醇、2-氨基-2-甲基-丙醇、1,1-二甲基双胍、苯乙基双胍、二氨基胍、葡糖胺和二甲基氨基乙醇。
[42]根据上述[36]的药物，所述叶酸衍生物包括亚叶酸钙。
[43]根据上述[36]的药物，所述药物的剂型为儿童适用的剂型或成人适用的剂型。
[44]根据上述[43]的药物，所述儿童包括出生28天内的新生儿、1岁以内的婴儿、1～6岁的幼儿、以及6～18岁的儿童；
[45]根据上述[43]的药物，所述成人包括妊娠期的女性成年人、围产期的女性成年人和哺乳期的女性成年人。
[46]根据上述[36]的药物，所述药物的剂型包括胃肠道给药的剂型和非胃肠道给药的剂型。
[47]根据上述[36]的药物，所述药物的剂型包括胶囊剂、片剂、微囊制剂、注射剂、栓剂、喷雾剂、散剂、软胶囊剂、滴丸、蜜丸、丸剂、颗粒剂、蜜炼膏剂、缓控释制剂、口服液体制剂、注射剂、咀嚼片、口腔片、透皮贴剂和泡腾片。
[48]根据上述[36]的药物，所述叶酸或其衍生物在所述药物中的单位剂量为0.1mg～1g。
[49]根据上述[36]的药物，所述叶酸或其衍生物在所述药物中的单位剂量为0.1mg、0.2mg、0.3mg、0.4mg、0.5mg、0.6mg、0.7mg、0.8mg、0.9mg或1.0mg。
[50]根据上述[36]的药物，所述叶酸或其衍生物在所述药物中的单位剂量为0.2mg、0.4mg、0.8mg、或1.0mg。
[51]一种预防和/或治疗遗传性、感染性或过敏性疾病的方法，包括向受试者施用包括叶酸或其衍生物作为活性成分的药物，其中，所述遗传性疾病包括胆道闭锁，所述感染性疾病包括胆管炎、黄疸、新生儿感染、坏死性小肠结肠炎、细菌感染性疾病、病毒感染性疾病，所述过敏性疾病包括食物过敏。
[52]根据上述[51]的方法，所述胆道闭锁包括病毒引起的胆道闭锁、或胆汁淤积引起的胆道闭锁。
[53]根据上述[52]的方法，所述病毒引起的胆道闭锁还包括主要由巨细胞病毒引起的胆道闭锁、或主要由 轮状病毒引起的胆道闭锁。
[54]根据上述[51]的方法，所述胆道闭锁还表现为胆道闭锁引起的食物过敏、胆道闭锁引起的黄疸、胆道闭锁引起的胆管炎、胆道闭锁引起的肝脏疾病、胆道闭锁引起的肠道性疾病。
[55]根据上述[54]的方法，所述胆道闭锁引起的肝脏疾病包括胆道闭锁引起的肝脏功能损伤、和/或胆道闭锁引起的肝脏炎症性疾病。
[56]根据上述[54]的方法，所述胆道闭锁引起的肠道性疾病包括胆道闭锁引起的肠道性炎症性疾病。
[57]根据上述[51]的方法，所述黄疸包括病理性黄疸或婴儿期黄疸。
[58]根据上述[57]的方法，所述病理性黄疸包括病毒引起的病理性黄疸、或婴儿期病理性黄疸。
[59]根据上述[57]的方法，所述病理性黄疸还包括病毒引起的婴儿期病理性黄疸；或优选的，所述病毒引起的病理性黄疸包括巨细胞病毒性黄疸。
[60]根据上述[51]的方法，所述胆管炎包括细菌性胆管炎、病毒性胆管炎、胆道闭锁引起的胆管炎、胆道闭锁术后并发的胆管炎。
[61]根据上述[60]的方法，所述细菌性胆管炎还包括胆道闭锁引起的细菌性胆管炎、胆道闭锁术后并发的细菌性胆管炎。
[62]根据上述[51]的方法，所述新生儿感染性疾病还包括新生儿细菌感染疾病和新生儿病毒感染疾病、新生儿支原体或者新生儿衣原体感染；或所述新生儿感染性疾病包括新生儿肝炎综合征、新生儿败血症、新生儿肺炎、新生儿感染导致的胃肠道疾病。
[63]根据上述[62]的方法，所述新生儿病毒感染还包括RNA病毒和DNA病毒。
[64]根据上述[63]的方法，所述RNA病毒还包括反义RNA病毒、DNA病毒。
[65]根据上述[62]的方法，所述新生儿病毒感染还包括新生儿巨细胞病毒(CMV)感染、新生儿轮状病毒感染、新生儿疱疹病毒感染。
[66]根据上述[51]的方法，所述细菌感染性疾病包括大肠杆菌、金黄色葡萄球菌或溶血性链球菌。
[67]根据上述[51]的方法，所述病毒感染性疾病包括巨细胞病毒(CMV)感染、轮状病毒感染、疱疹病毒感染。
[68]根据上述[67]的方法，所述病毒性感染疾病还包括病毒性腹泻。
[69]根据上述[51]的方法，所述食物过敏还表现为食物过敏导致的皮肤疾病、食物过敏导致的胃肠道疾病、食物过敏导致的呼吸道疾病。
[70]根据上述[69]的方法，所述食物过敏导致的胃肠道疾病包括食物过敏导致的炎症性肠病。
[71]根据上述[51]的方法，所述叶酸衍生物包括叶酸在药学上可以接受的盐、酯、水合物、溶剂化物、多晶型物、互变异构体、前药和叶酸功能等价物。
[72]根据上述[51]的方法，所述叶酸衍生物包括(a1)～(a2)中任一种：
(a1)亚叶酸、二氢叶酸、四氢叶酸、5-甲基四氢叶酸、5,10-亚甲基四氢叶酸、5,10-亚甲基四氢叶酸、5,10-甲酰亚氨基四氢叶酸、5-甲酰四氢叶酸、10-甲酰四氢叶酸和10-甲基四氢叶酸；
(a2)为(a1)的药学上可接受的盐。
[73]根据上述[71]-[72]的方法，所述药学上的盐包括钙、钠、锌、精氨酸、胆碱、乙酰胆碱、N甲基氨基乙醇、2-氨基-2-甲基-丙醇、1,1-二甲基双胍、苯乙基双胍、二氨基胍、葡糖胺和二甲基氨基乙醇。
[74]根据上述[51]的方法，所述叶酸衍生物包括亚叶酸钙。
[75]根据上述[51]的方法，所述治疗方法还包含向受试者施加其他活性成分，所述其他活性成分包含维生素B、维生素C、烟酰胺、氧化锌、维生素A、维生素E、同型半胱氨酸、谷胱甘肽和牛磺酸中的至少一种。
[76]根据上述[75]的方法，所述维生素B包含维生素B1、维生素B2、维生素B3、维生素B5、维生素B6、维生素B7和维生素B12中的至少一种。
[77]根据上述[51]的方法，所述药物包括叶酸、及维生素B6和B12中的至少一种；或，所述药物包括：叶酸或其衍生物；和维生素B6、维生素B12、维生素A、维生素E、维生素C、维生素D、维生素K、烟酰胺、氧化锌、牛磺酸中的至少一种；或，所述药物包括叶酸或其衍生物，和烟酰胺；或，所述药物包括叶酸或其衍生物，和维生素E；或，所述药物包括叶酸或其衍生物，和维生素A和维生素E。
[78]根据上述[51]的方法，所述药物的剂型为儿童适用的剂型或成人适用的剂型。
[79]根据上述[78]的方法，所述儿童包括出生28天内的新生儿、1岁以内的婴儿、1～6岁的幼儿、以及6～18岁的儿童。
[80]根据上述[78]的方法，所述成人包括妊娠期的女性成年人、围产期的女性成年人和哺乳期的女性成年人。
[81]根据上述[51]的方法，所述药物的剂型包括胃肠道给药的剂型和非胃肠道给药的剂型。
[82]根据上述[51]的方法，所述药物的剂型包括胶囊剂、片剂、微囊制剂、注射剂、栓剂、喷雾剂、散剂、软胶囊剂、滴丸、蜜丸、丸剂、颗粒剂、蜜炼膏剂、缓控释制剂、口服液体制剂、注射剂、咀嚼片、口腔片、透皮贴剂和泡腾片。
[83]根据上述[51]的方法，所述叶酸或其衍生物在所述药物中的单位剂量为0.1mg～1g。
[84]根据上述[51]的方法，所述叶酸或其衍生物在所述药物中的单位剂量为0.1mg、0.2mg、0.3mg、0.4mg、0.5mg、0.6mg、0.7mg、0.8mg、0.9mg或1.0mg。
[85]根据上述[51]的方法，所述叶酸或其衍生物在所述药物中的单位剂量为0.2mg、0.4mg、0.8mg、或1.0mg。
[86]胆道闭锁的诊断或辅助诊断标志物，所述诊断或辅助诊断标志物包含：
a)叶酸；以及
b)S100a8、S100a9、Nox2、IFN-γ中的至少一种。
[87]根据[86]的标志物，所述S100a8、S100a9、Nox2和IFN-γ包括蛋白质和/或mRNA。
[88]根据[86]的标志物，所述诊断或辅助诊断标志物来自体液、血液、组织、细胞或排泄物。
[89]根据[88]的标志物，所述组织包括肝脏、肠道。
[90]根据[88]的标志物，所述排泄物包括粪便、尿液。
[91]根据[88]的标志物，所述血液为全血、血清或血浆、干血斑。
[92]检测标志物的试剂在制备诊断或辅助诊断胆道闭锁的产品中的应用：所述标志物包含叶酸、S100a8、S100a9、Nox2、IFN-γ中的至少一种。
[93]根据[92]的应用，所述S100a8、S100a9、Nox2、IFN-γ包括蛋白质和/或mRNA。
[94]根据[92]的应用，所述产品包括试剂、试剂盒、基因芯片和蛋白质芯片。
[95]根据[92]的应用，所述产品用于执行以下检测方法中的至少一种：
(d1)比色法；(d2)化学发光法；(d3)原子吸收分光光度法；(d4)聚合酶链反应；(d5)微数字聚合酶链反应；(d6)荧光聚合酶链反应；(d7)环介导等温扩增反应；(d8)酶联免疫吸附测定法；(d9)核苷酸序列测序法；(d10)氨基酸序列测序法；(d11)变性梯度凝胶电泳；(d12)核酸分型芯片检测；(d13)高效液相色谱法；(d14)原位杂交；(d15)生物质谱法；(d16)高分辨率溶解曲线分析；(d17)单链构象异构多态分析；(d18)探针扩增阻滞突变系统分析。
[96]根据[92]的应用，当标志物为S100a8、S100a9、Nox2和IFN-γ的mRNA时，所述产品含有检测所述标志物和/或内参mRNA的引物、和/或检测探针。
[97]根据[92]的应用，当标志物为S100a8mRNA，所述产品包括检测S100a8mRNA的引物，所述引物的核苷酸序列如SEQ ID NO.1和SEQ ID NO.2所示。
[98]根据[92]的应用，当标志物为S100a9mRNA，所述产品包括检测S100a9mRNA的引物，所述引物的核苷酸序列如SEQ ID NO.3和SEQ ID NO.4所示。
[99]根据[92]的应用，当标志物为IFN-γFmRNA，所述产品包括检测IFN-γmRNA的引物，所述引物的核苷酸序列如SEQ ID NO.5和SEQ ID NO.6所示。
[100]根据[92]的应用，当标志物为Nox2 mRNA，所述产品包括检测Nox2 mRNA的引物，所述引物的核苷酸序列如SEQ ID NO.7和SEQ ID NO.8所示。
[101]根据[92]的应用，当待测对象满足(c1)～(c5)中至少一项，则诊断为胆道闭锁疾病患者；
(c1)叶酸相对于参考水平显著降低；
(c2)S100a8相对于参考水平显著升高；
(c3)S100a9相对于参考水平显著升高；
(c4)IFN-γ相对于参考水平显著升高；
(c5)Nox2相对于参考水平显著降低；
所述参考水平为未患有胆道闭锁疾病的同年龄段受试者的水平。
[102]根据[101]的应用，所述同年龄段受试者为健康人或胆总管囊肿患者。
[103]根据[92]的应用，所述标志物来自体液、血液、组织、细胞或排泄物。
[104]根据[103]的应用，所述组织包括肝脏、肠道。
[105]根据[103]的应用，所述排泄物包括粪便、尿液。
[106]根据[103]的应用，所述血液为全血、血清或血浆、干血斑。
[107]根据[103]的应用，所述待测对象为成人或儿童。
[108]根据[107]的应用，所述儿童包括出生28天内的新生儿、1岁以内的婴儿、1～6岁的幼儿、以及6～8岁的儿童。
[109]根据[107]的应用，所述成人选自妊娠期的女性成年人、围产期的女性成年人或哺乳期的女性。
[110]一种试剂盒，包含检测标志物的试剂；所述标志物包含叶酸、S100a8、S100a9、Nox2、IFN-γ中的至少一种。
[111]根据[110]的试剂盒，所述标志物包含：
a)叶酸；以及
b)S100a8、S100a9、Nox2、IFN-γ中的至少一种。
[112]根据[110]的试剂盒，当检测标志物为S100a8、S100a9、Nox2和IFN-γ的mRNA时，所述产品含有检测所述标志物和/或内参mRNA的引物、和/或检测探针。
[113]根据[110]的试剂盒，当检测标志物为S100a8、S100a9、Nox2和IFN-γ的mRNA时，所述产品还包含：样品的处理试剂，包括但不限于样品裂解试剂、样品纯化试剂和/或样品核酸提取试剂。
[114]根据[110]的试剂盒，当检测标志物为S100a8、S100a9、Nox2和IFN-γ的mRNA时，所述产品还包含：RNA提取试剂、dNTP、DNA聚合酶、双链特异性荧光染料以及水中的一种或多种。
[115]根据[110]的试剂盒，当检测标志物为S100a8mRNA，所述产品包括检测S100a8mRNA的引物，所述引物的核苷酸序列如SEQ ID NO.1和SEQ ID NO.2所示。
[116]根据[110]的试剂盒，当检测标志物为S100a9mRNA，所述产品包括检测S100a9mRNA的引物，所述引物的核苷酸序列如SEQ ID NO.3和SEQ ID NO.4所示。
[117]根据[110]的试剂盒，当检测标志物为IFN-γmRNA，所述产品包括检测IFN-γmRNA的引物，所述引物的核苷酸序列如SEQ ID NO.5和SEQ ID NO.6所示。
[118]根据[110]的试剂盒，当检测标志物为Nox2 mRNA，所述产品包括检测Nox2 mRNA的引物，所述引物的核苷酸序列如SEQ ID NO.7和SEQ ID NO.8所示。
[119]根据[110]的试剂盒，所述产品的检测样本或受试样本来自待测对象的来自体液、血液、组织、细胞或排泄物；进一步优选地，所述标志物来自血液、组织或排泄物。
[120]根据[119]的试剂盒，所述组织包括肝脏、肠道。
[121]根据[119]的试剂盒，所述排泄物包括粪便、尿液。
[122]根据[119]的试剂盒，所述血液为全血、血清或血浆、干血斑；进一步为血清、干血斑。
[123]根据[110]的试剂盒，所述待测对象为成人或儿童。
[124]根据[123]的试剂盒，所述儿童包括出生28天内的新生儿、1岁以内的婴儿、1～6岁的幼儿、以及6～8岁的儿童。
[125]根据[123]的试剂盒，所述成人选自妊娠期的女性成年人、围产期的女性成年人或哺乳期的女性成年人。
[126]根据[110]的试剂盒，当待测对象满足(c1)～(c5)中至少一项，则诊断为胆道闭锁疾病患者；
(c1)叶酸相对于参考水平显著降低；
(c2)S100a8相对于参考水平显著升高；
(c3)S100a9相对于参考水平显著升高；
(c4)IFN-γ相对于参考水平显著升高；
(c5)Nox2相对于参考水平显著降低；
所述参考水平为未患有胆道闭锁疾病的同年龄段受试者的水平。
[127]根据[126]的试剂盒，所述同年龄段受试者为健康人或胆总管囊肿患者。
[128]一种诊断或辅助诊断胆道闭锁疾病的方法，所述方法包含检测标志物：叶酸、S100a8、S100a9、Nox2、IFN-γ中的至少一种的水平和/或活性。
[129]根据[128]的方法，所述方法包含检测
a)叶酸；以及
b)S100a8、S100a9、Nox2、IFN-γ中的至少一种的水平和/或活性。
[130]根据[128]的方法，所述方法的检测样本或受试样本来自待测对象的来自体液、血液、组织、细胞或排泄物；进一步优选地，所述标志物来自血液、组织或排泄物。
[131]根据[130]的方法，所述组织包括肝脏、肠道。
[132]根据[130]的方法，所述排泄物包括粪便、尿液。
[133]根据[130]的方法，所述血液为全血、血清或血浆、干血斑；进一步为血清、干血斑。
[134]根据[128]的方法，所述待测对象为成人或儿童。
[135]根据[134]的方法，所述儿童包括出生28天内的新生儿、1岁以内的婴儿、1～6岁的幼儿、以及6～8岁的儿童。
[136]根据[134]的方法，所述成人选自妊娠期的女性成年人、围产期的女性成年人或哺乳期的女性成年人。
[137]根据[128]的方法，当待测对象满足(c1)～(c5)中至少一项，则诊断为胆道闭锁疾病患者；
(c1)叶酸相对于参考水平显著降低；
(c2)S100a8相对于参考水平显著升高；
(c3)S100a9相对于参考水平显著升高；
(c4)IFN-γ相对于参考水平显著升高；
(c5)Nox2相对于参考水平显著降低；
所述参考水平为未患有胆道闭锁疾病的同年龄段受试者的水平。
[138]根据[137]的方法，所述同年龄段受试者为健康人或胆总管囊肿患者。
[139]根据[128]的方法，当检测标志物为S100a8、S100a9、Nox2和IFN-γ的mRNA时，所述检测方法还包括使用所述标志物和/或内参mRNA的引物、和/或检测探针。
[140]根据[128]的方法，当检测标志物为S100a8、S100a9、Nox2和IFN-γ的mRNA时，所述检测方法还 包括使用：样品的处理试剂，包括但不限于样品裂解试剂、样品纯化试剂和/或样品核酸提取试剂。
[141]根据[128]的方法，当检测标志物为S100a8、S100a9、Nox2和IFN-γ的mRNA时，所述检测方法还包括使用：RNA提取试剂、dNTP、DNA聚合酶、双链特异性荧光染料以及水中的一种或多种。
[142]根据[128]的方法，当检测标志物为S100a8mRNA，所述检测方法还包括使用S100a8mRNA的引物，所述引物的核苷酸序列如SEQ ID NO.1和SEQ ID NO.2所示。
[143]根据[128]的方法，当检测标志物为S100a9mRNA，所述检测方法还包括使用检测S100a9mRNA的引物，所述引物的核苷酸序列如SEQ ID NO.3和SEQ ID NO.4所示。
[144]根据[128]的方法，当检测标志物为IFN-γmRNA，所述检测方法还包括使用检测IFN-γmRNA的引物，所述引物的核苷酸序列如SEQ ID NO.5和SEQ ID NO.6所示。
[145]根据[128]的方法，当检测标志物为Nox2 mRNA，所述检测方法还包括使用检测Nox2 mRNA的引物，所述引物的核苷酸序列如SEQ ID NO.7和SEQ ID NO.8所示。
本发明的有益效果是：
1.本发明实施例表明，采用叶酸干预RRV致胆道闭锁模型小鼠后，非常有效地控制了胆道闭锁的发生和发展。因此，本发明应用了一种人每天都需要的安全性高的营养物质——叶酸或其衍生物，来防治胆道闭锁及其相关度高的疾病胆管炎，叶酸或其衍生物(例如其药用盐)作为一种安全的水溶性维生素，可直接应用、添加至营养食品或营养制剂中应用于备孕、怀孕、哺乳期妇女和/或新生婴儿，从而从根本上防治了胆道闭锁的发生、发展和预后，且毒副作用少。
2.本发明的实施例证明了，补充叶酸还能抑制肠道炎症因子的表达、降低肠道铁的含量、纠正肠道菌群紊乱；从而，本发明提供了一种经济、安全、有效的调节肠道菌群平衡、防治炎症性肠病等肠炎性疾病的组合物，其活性成分为叶酸或其衍生物，为广大胃肠道疾病患者提供了另一种防治胃肠道急/慢性、炎症/非炎症疾病的可行性方案。
3.本发明实施例表明，在病毒感染性胆道闭锁小鼠模型中使用叶酸，可以显著降低小鼠的黄疸率、改善小鼠肝脏功能、抑制肝脏或肠道细胞毒性细胞因子IFN-γ的表达，同时，还能抑制肠道炎症因子的表达、降低肠道铁的含量、纠正肠道菌群紊乱。因此，本发明提供了一种能同时作用于胆、肝、肠消化道的安全活性成分——叶酸，从而，本发明除了可防治胆道闭锁、菌群紊乱外，还可通过其作用靶点的多样性，达到对肝纤维化、肝肠循环障碍、病毒感染等疾病及相对于胆道闭锁轻微的疾病——胆管炎的防治作用。
4.本发明的实施例证明了，在BA小鼠中，叶酸可以抑制肠道和/或肝脏中炎症细胞因子S100a8、S100a9和IFN-γ的表达以及疾病中出现的铁离子过载情形，促进髓源性抑制细胞特征性转录因子Nox2的表达，并证明了：叶酸、S100a8、S100a9、IFN-γ、Nox2中的至少一种可以作为胆道闭锁的诊断或辅助诊断的标志物。
5.本发明还证明了：叶酸还可以降低胆道闭锁患者的总胆红素浓度和直接胆红素浓度，达到预防和/或治疗胆管炎的效果。同时，补充叶酸还可以达到纠正全身铁超载和管腔铁缺乏、抑制小肠嗜酸性粒细胞浸润、抑制IgG-Ro/SSA自身抗体的产生，增加IgM/IgG4比值，同时，延长自身免疫期和改善肝脏功能；本发明还证明了：补充亚叶酸钙(CF)可降低肝脏中的INFβ、ROS的含量，降低黄疸发生率并改善新生儿生长；提高小肠中Dhfr、Mthfr、Nrf2、Slc40a1的表达量，从而使叶酸代谢、氧化应激和铁稳态得到恢复；降低肝脏中CD4 +T细胞、CD8 +T细胞中的IFN-γ含量、浆细胞中IgG1含量；降低肝脏门管区周围淋巴细胞浸润；增加肝小叶结构的完整性；使肠上皮细胞的气球样变性得到恢复，达到治疗胆道闭锁的效果；通过补充叶酸或其衍生物(亚叶酸钙)可以预防和/或治疗细菌和/或病毒感染，从而达到预防和/或治疗新生儿感染疾病的效果。
因此，添加叶酸或其衍生物的营养食品和/或制剂和/或药物是一种安全、有效、经济、可行的防治胆道闭锁、胆管炎、肝纤维化、肝肠循环障碍、炎症性肠病、病毒性感染，调节肠道菌群平衡、新生儿感染疾病的方法。
附图说明
图1是胆道闭锁(BA)和胆总管囊肿(CC)患者中叶酸含量结果图：其中，A是胆道闭锁(BA)和胆总管囊肿(CC)患者血清中叶酸水平结果图；B是叶酸在区分BA与非BA受试者(胆总管囊肿(CC)患者)的预测性能的接受者操作特征(ROC)曲线图。
图2是叶酸对胆道闭锁小鼠模型的预防和治疗效果的结果图：其中，A是BA模型小鼠的治疗方案示意图；B是叶酸对胆道闭锁小鼠模型体重影响的结果图；C是叶酸对胆道闭锁小鼠模型的黄疸率影响的结果图；D是叶酸对胆道闭锁小鼠模型肝脏的坏死灶和淋巴细胞浸润情况影响的结果图；E是叶酸对胆道闭锁小鼠模型肝脏中CD4 +T细胞中的IFN-γ含量影响的结果图。
图3是叶酸对胆道闭锁小鼠模型肠道上皮的影响图。
图4是叶酸对胆道闭锁小鼠模型肠道绒毛的影响图。
图5是叶酸对胆道闭锁小鼠模型中炎症因子影响的结果图。
图6是叶酸对病毒或细菌感染所引起的I型干扰素通路的过度激活的影响图。
图7是亚叶酸钙对胆道闭锁小鼠模型的预防和治疗效果的结果图：其中，A是亚叶酸钙对胆道闭锁小鼠模型体重影响的结果图；B是亚叶酸钙对胆道闭锁小鼠模型的肝脏门管区周围淋巴细胞浸润、肝小叶结构的完整性、以及肠上皮细胞的气球样变性的影响的结果图；C是亚叶酸钙对胆道闭锁小鼠模型的黄疸率影响的结果图；D是亚叶酸钙对胆道闭锁小鼠模型肝脏中INFβ、ROS的含量影响的结果图；E是亚叶酸钙对胆道闭锁小鼠模型小肠中Dhfr、Mthfr、Nrf2、Slc40a1的表达量；F是亚叶酸钙对胆道闭锁小鼠模型的肝脏中CD4 +T细胞、CD8 +T细胞中的IFN-γ含量、浆细胞中IgG1含量的影响图。
图8是叶酸对胆道闭锁婴儿胆管炎的发病率的影响图：其中，A是BA婴儿的治疗方案示意图；B是BA婴儿的人口学和临床特征图；C是叶酸对胆道闭锁婴儿胆管炎的发病率的影响的汇总图。
图9是叶酸对胆道闭锁婴儿胆管炎的发病的影响图：其中，A是胆道闭锁婴儿接收叶酸治疗后不同时间内胆管炎的发病率；B是叶酸对胆道闭锁婴儿总胆红素浓度影响图；C是叶酸对胆道闭锁婴儿直接胆红素浓度影响图。
图10是叶酸治疗胆道闭锁机制的研究的结果图：其中，A是基于BD Rhapsody“M”平台的单细胞测序实验设计示意图；B是叶酸对肠细胞的分布和比例的影响图；C是叶酸治疗胆道闭锁机制的图形摘要。
图11是叶酸对肠细胞基因表达以及血浆中BH4和肝脏活检中HCY的浓度的影响图：其中，A是叶酸对指示基因在肠细胞亚群中的表达的影响图；B是叶酸对肠细胞SLC11A2、SLC40A1和SLC46A1基因表达的影响图；C是叶酸对血浆中BH4和肝脏活检中HCY的浓度的影响图。
图12是叶酸对铁浓度、肝脏中Ro/SSA IgG、Ro/SSA IgM、IgM/IgG比值的影响图：其中，A是叶酸对铁浓度的影响图；B是叶酸对肝脏中Ro/SSA IgG、Ro/SSA IgM、IgM/IgG比值的影响图。
图13是叶酸抑制胆道闭锁和胆囊肿患者食物过敏发生的结果图：其中，A是胆道闭锁术后和胆囊肿患者术后食物过敏发生比例调查结果图；B是服用叶酸7天后胆道闭锁术中患者肠道上参与食物过敏的嗜酸性粒细胞数量显著减少的结果图。
具体实施方式
以下结合具体的实施例及附图对本发明的内容作进一步详细的说明。
应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。
现将详细地提供本发明实施方式的参考，其一个或多个实例描述于下文。提供每一实例作为解释而非限制本发明。实际上，对本领域技术人员而言，显而易见的是，可以对本发明进行多种修改和变化而不背离本发明的范围或精神。例如，作为一个实施方式的部分而说明或描述的特征可以用于另一实施方式中，来产生更进一步的实施方式。
因此，旨在本发明覆盖落入所附权利要求的范围及其等同范围中的此类修改和变化。本发明的其它对象、特征和方面公开于以下详细描述中或从中是显而易见的。本领域普通技术人员应理解本讨论仅是示例性实施方式的描述，而非意在限制本发明更广阔的方面。
对于本发明提及的“剂量”，本领域技术人员应当理解，所述药物或组合物应包含至少0.1mg叶酸。一般正常人叶酸的需求量为0.2～0.4mg/天，孕妇和母乳为0.4mg/天。而出于疾病治疗的目的，则需要更高数量的叶酸，例如0.8mg～1g/天。为了方便给药，药物优选是单位制剂，即满足一次给药所需活性成分的制剂。常见的单位制剂如一单位(片)片剂、一单位(针)针剂或粉针剂等，其中活性成分的为一次给药所需的量。一次给药所需的量可以方便地计算受试者的体重和该受试者一次用药所需单位体重剂量(在本文中简称为“剂量”)的乘积得到。例如，在制备药物的过程中，通常将成人的体重被定为60kg，可以用该体重值来计算。不同受试者的单位体重剂量可以通过等效剂量换算关系来计算。例如，根据的本领域普通技术人员所公知的实验动物与人的等效剂量换算关系(通常可参见FDA、SFDA等药品管理机构的指导意见，也可参见“黄继汉等.药理试验中动物间和动物与人体间的等效剂量换算.中国临床药理学与治疗学，2004Sep；9(9)：1069-1072”)可从实验动物的剂量推导出人的有效剂量。本发明的活性成分叶酸或其衍生物(例如其可药用盐)可给患者每日剂量0.8mg～1g/天，给药量具体可根据患者性别、体重、年龄、症状的程度等因素实际情况进行调整。
对于本发明提及的“叶酸”，本领域技术人员应当理解，为叶酸(PTEROYLMONOGLUTAMATE)，或包括其所有功能等价物，以及其衍生物，一种或多种叶基聚谷氨酸盐，其中叶酸或多谷氨酸的蝶呤部分的吡嗪环被还原成二氢叶酸或四氢叶酸的化合物，或所有上述化合物的衍生物，其中N-5或N-10位在各种氧化水平上携带一个碳单元，或其药学上相容的盐或其两种或多种的组合。特别是指叶酸，以游离叶酸(蝶酰谷氨酸)或亚叶酸(甲酰四氢叶酸)形式存在。此外，叶酸的等价物或衍生物可选：亚叶酸，二氢叶酸，四氢叶酸，5-甲基四氢叶酸，5,10-亚甲基四氢叶酸，5,10-亚甲基四氢叶酸，5,10-甲酰亚氨基四氢叶酸，5-甲酰四氢叶酸(隐叶素)，10-甲酰四氢叶酸，10-甲基四氢叶酸，其药学上可接受的盐，或其两种或多种的组合。
对于本发明提及的“维生素B族”，本领域技术人员应当理解，维生素B族包括维生素B1(硫胺素)、维生素B2(核黄素)、维生素B3(烟酸)、维生素B5(泛酸)、维生素B6(吡哆醇)、维生素B12(氰钴胺)、维生素B7(生物素)等，是促进叶酸吸收的有效成分。
对于本发明提及的“维生素C”，具有增强叶酸疗效的作用。
对于本发明中提及的“维生素A”，是一种脂溶性维生素，有机化合物，化学式是C 20H 30O，对热、酸、碱稳定，易被氧化，紫外线可促进其氧化破坏。维生素A包括A1及A2，A1即视黄醇。维生素A2即3-脱氢视黄醇。维生素A有促进生长、繁殖，维持骨骼、上皮组织、视力和粘膜上皮正常分泌等多种生理功能，维生素A及其类似物有阻止癌前期病变的作用。
同型半胱氨酸，(又称高半胱氨酸，血浆同型半胱氨酸，homocysteine,Hcy)是蛋氨酸代谢过程中的中间产物，其结构式为HSCH 2(NH 2)CO 2H。血浆中存在氧化型和还原型HCY两种形式，氧化型含二硫基，包括同型胱氨酸和胱氨酸；还原型含硫基，包括同型半胱氨酸及半胱氨酸。
对于本发明提及的“牛磺酸”，具有保肝、护肝作用，可以促进胃肠功能，增加人体免疫力，提高机体抵抗力，提高抗病能力，本领域技术人应可以理解，将其应用与胆道闭锁的治疗药物中，可以缩短具有肝损伤的胆道闭锁疾病的治疗病程。
对于本发明提及的“胆道闭锁”，本领域技术人员应当理解，胆道闭锁(biliary atresia，BA)是婴儿期常见的严重肝胆系统疾病之一，以肝内、外胆管进行性炎症和纤维化为特征；如不及时治疗，晚期会出现胆汁性肝硬化、门静脉高压、肝衰竭，是一种病因不明的波及肝内、外胆管闭塞性病变，导致胆汁淤积及进行性肝纤维化直至肝硬化并危及患儿生命的疾病。临床表现为：(1)生后黄疸延迟消退(足月儿大于2周，早产儿大于3周)，或消退后再次出现，并持续性加重；(2)粪便颜色逐渐变浅至白陶土色，尿色加深至浓茶色； (3)腹部膨隆，肝脾肿大，腹壁静脉曲张等；(4)由于脂溶性维生素吸收障碍导致营养不良或生长发育迟缓。
对于本发明提及的“肝肠循环障碍”，本领域技术人员应当理解，所述肝肠循环障碍所导致的疾病包括高胆固醇血症、胆固醇结石、黄疸。
对于本发明提及的炎症性肠病”，本领域技术人员应当理解，炎症性肠病(inflammatory bowel disease，IBD)为累及回肠、直肠、结肠的一种特发性肠道炎症性疾病。临床表现腹泻、腹痛，甚至可有血便。本病包括溃疡性结肠炎(UC)和克罗恩病(CD)。溃疡性结肠炎是结肠黏膜层和黏膜下层连续性炎症，疾病通常先累及直肠，逐渐向全结肠蔓延,克罗恩病可累及全消化道，为非连续性全层炎症，最常累及部位为末端回肠、结肠和肛周。
对于本发明提及的病毒性感染”，本领域技术人员应当理解，病毒感染(viral infection)是指病毒通过多种途径侵入机体，并在易感的宿主细胞中增殖的过程。人类病毒是指能感染人体或对人有致病作用的病毒。病毒感染的实质是病毒与机体、病毒与易感细胞相互作用的过程。病毒感染常因病毒种类、机体状态不同产生轻重不一的损伤或病毒性疾病。病毒致病是由侵入宿主、感染细胞开始的，致病作用表现在人整体和细胞两个方面。
对于本发明提及的肠道菌群紊乱”，本领域技术人员应当理解，健康人的肠道中会有稳定而均衡的菌群存在，维护肠道环境的正常，一旦这种肠道环境被破坏就会引起肠道菌群紊乱，这种疾病必然会引起机体消化系统的病症。肠道菌群紊乱容易导致的疾病包括但不限于：
1.白色念珠菌性肠炎
是肠道菌群紊乱症最常见的一种。多见于瘦弱的婴儿、消化不良、营养不良、糖尿病、恶性肿瘤、长期应用抗生素或激素的患者。
2.葡萄球菌性肠炎
多见于长期应用抗生素(四环素类、氨苄青霉素等)、肾上腺皮质激素和进行肠道手术的老年患者或慢性病患者。
3.产气荚膜杆菌性急性坏死性肠炎
产气荚膜杆菌所产生的β霉素可引起急性坏死性肿瘤、消耗性疾病、以及使用抗生素、皮质激素等情况下最易发生感染。
4.坏死性小肠结肠炎和败血症
为一种获得性疾病，主要在早产儿或患病的新生儿中发生(新生儿坏死性小肠结肠炎，为NEC)，其特征为粘膜甚至为肠深层的坏死,最常发生在回肠末端,结肠和近端小肠很少受累，流行病学的研究已证实一些集中散发病例与特殊的病原菌在肠道中的定植有关(如克雷白菌、大肠杆菌、凝固酶阴性的葡萄球菌)。肠坏死从粘膜层开始，逐渐累及肠壁全层，导致穿孔，并且1/3的新生儿继发发生败血症。
5.绿脓杆菌肠道感染
绿脓杆菌为条件致病菌，常为继发感染，在婴幼儿、老人、某些恶性肿瘤、消耗性疾病、以及使用抗生素、皮质激素等情况下最易发生感染。
6.变形杆菌肠道感染
变形杆菌在一定条件下可为条件致病菌，如普通杆菌、奇异杆菌、摩根氏变形杆菌均可引起食物中毒，无恒变形杆菌可引起婴幼儿夏季腹泻。
7.肺炎杆菌肠道感染
当机体抵抗力降低或其他原因，正常寄生在肠道的肺炎杆菌可引起感染，特别是小儿的严重腹泻。
肠道菌群紊乱症引起肠道的炎症是极为普遍的，同时，腹泻也是肠道菌群紊乱症最为主要的临床症状，如果腹泻症状严重，还会引起机体脱水、血压下降等症状，同时如果不及时治疗还可能引起呼吸道系统或泌 尿系统的疾病。
对于本发明提及的妊娠期”，本领域技术人员应当理解，妊娠期是指受孕后至分娩前的生理时期，亦称怀孕期。自成熟卵受精后至胎儿娩出，一般为266天左右。为便于计算，妊娠通常是从末次月经第一天算起，足月妊娠约为280天(40周)。在妊娠期间母体的新陈代谢、消化系统、呼吸系统、血管系统、神经系统、内分泌系统、生殖系统、骨关节韧带及乳房均发生相应的改变。妊娠期全过程共分为3个时期：妊娠13周末以前称早期妊娠；第14～27周末称中期妊娠；第28周及其后称晚期妊娠。
对于本发明提及的“围产期”，本领域技术人员应当理解，围产期是指怀孕28周到产后一周这一分娩前后的时期。
对于本发明提及的“哺乳期”，本领域技术人员应当理解，哺乳期是指产后产妇用自己的乳汁喂养婴儿的时期，即开始哺乳到停止哺乳的这段时间，一般长约10个月至2年左右。
对于本发明提及的“诊断或辅助诊断标记物”，生物标志物(Biomarker)是指可以标记系统、器官、组织、细胞及亚细胞结构或功能的改变或可能发生的改变的生化指标，具有非常广泛的用途。生物标志物可用于疾病诊断、判断疾病分期或者用来评价新药或新疗法在目标人群中的安全性及有效性。本领域技术人员应当理解，对于疾病研究，生物标志物一般是指可供客观测定和评价的一个普通生理或病理或治疗过程中的某种特征性的生化指标，通过对它的测定可以获知机体当前所处的生物学过程中的进程。检查一种疾病特异性的生物标志物，对于疾病的鉴定、早期诊断及预防、治疗过程中的监控可能起到帮助作用。
对于本发明提及的γ干扰素，本领域技术人员应当理解，γ干扰素又称γ-IFN或免疫干扰素是由有丝分裂原刺激T淋巴细胞产生。干扰素是一种高效的抗病毒生物活性物质，又是一种具有广泛免疫调节作用的淋巴因子。
对于本发明提及的“剂型”，本领域技术人员应当理解，药物剂型的分类：
(一)按给药途径分类
1.经胃肠道给药剂型
2.非经胃肠道给药剂型
(1)注射给药剂型：如各种粉针剂、水针剂。
(2)呼吸道给药剂型：如盐酸异丙肾上腺素气雾剂。
(3)皮肤给药剂型：如硼酸洗剂。
(4)粘膜给药剂型：如红霉素眼药膏。
(5)腔道给药剂型：如用于直肠、阴道、尿道的各种栓剂。
(二)按分散系统分类
1.溶液型
2.胶体溶液型
3.乳剂型
4.混悬型
5.气体分散型
6.微粒分散型
7.固体分散型
(三)按形态分类
液体剂型，气体剂型，固体剂型和半固体剂型。
对于本发明提及的“辅料”，本领域技术人员应当理解，药用辅料是指生物药品和调剂处方时所用的赋形剂与附加剂，即除主要活性成分以外的一切物料的总称。在注射剂制备过程中，为确保安全、有效、稳定， 除主药和溶剂之外常加入辅料以增加溶解度、提高稳定性等。药用辅料除了赋形、充当载体、提高稳定性外，还具有增溶、助溶、缓控释等重要功能，是可能会影响到药品的质量、安全性和有效性的重要成分。是指在制剂处方设计时，为解决制剂的成型性、有效性、稳定性、安全性加入处方中除主药以外的一切药用物料的统称。药物制剂处方设计过程实质是依据药物特性与剂型要求，筛选与应用药用辅料的过程。
药用辅料是药物制剂的基础材料和重要组成部分，是保证药物制剂生产和发展的物质基础，在制剂剂型和生产中起着关键的作用。它不仅赋予药物一定剂型。而且与提高药物的疗效、降低不良反应有很大的关系，其质量可靠性和多样性是保证剂型和制剂先进性的基础。按用途可分为溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、黏合剂、崩解剂、填充剂、润滑剂、润湿剂、渗透压调节剂、稳定剂、助流剂、矫味剂、防腐剂、助悬剂、包衣材料、芳香剂、抗黏合剂、整合剂、渗透促进剂、pH值调节剂、缓冲剂、增塑剂、表面活性剂、发泡剂、消泡剂、增稠剂、包合剂、保湿剂、吸收剂、稀释剂、絮凝剂与反絮凝剂、助滤剂、释放阻滞剂等。
对于本发明提及的“S100a8”、“S100a9”，本领域技术人员应当理解，S100a8、S100a9编码S100A8(S100 Calcium Binding Protein A8)和S100A9(S100 Calcium Binding Protein A9)钙结合蛋白，常形成S100A8/A9异二聚体。研究表明S100A8和S100A9与慢性炎症性疾病及多种肿瘤的许多病理过程密切相关。S100A8和S100A9蛋白主要通过结合并活化Toll样受体和糖基化终产物受体从而介导细胞内的炎症信号传导等途径,在炎症中发挥重要的调节作用。
对于本发明提及的髓源性抑制细胞(myeloid derived suppressive cells,MDSC)，是一群在病理条件(例如肿瘤、自身免疫疾病、感染等)下产生的具有显著抑制功能的天然免疫细胞，其分化发生异常,导致MDSC累积,多条信号通路的调控和多种细胞因子受到影响，Nox2便是其中之一。“Nox2”，是指Nox2编码NADPH氧化酶2，是NADPH氧化酶家族中的重要一员，在炎症中起到重要的调节作用。
对于本发明中所述的基因/蛋白，本领域技术人员知晓，基因和蛋白的表达是对应的，因此，在证明基因具有一种功能时候，也说明其对应的蛋白也具有相应功能；反之亦然。
对于本发明提及的“儿童”，本领域技术人员应当理解，儿童是指18岁以下的任何人，优选地，其为出生28天内的新生儿、1岁以内的婴儿、1-6岁的幼儿、以及6岁以上18岁以下的儿童。
对于本发明提及的“成人”，本领域技术人员应当理解，成人是指18岁以上的任何人，优选地，所述成人为妊娠期的女性成年人、围产期的女性成年人或哺乳期的女性成年人。
除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。除非特别说明，以下实施例所用试剂和材料均为市购。
除非另有说明，本发明采用的免疫学、生物化学、化学、分子生物学、微生物学、细胞生物学、基因组学和重组DNA等是本领域的常规技能。
下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。本实施例中所使用的材料、试剂等，如无特别说明，为从商业途径得到的试剂和材料。
实施例1.胆道闭锁患者标本的获取
为了明确BA疾病患者疾病发生发展的原因，本发明收集临床肠道、肝脏以及血液标本，检测代谢相关指标。
1.临床标本以及数据的收集：
胆道闭锁及对照组胆总管囊肿患儿的病史，尤其是肝脏损伤相关实验室检测指标从医院的电子医疗档案系统中获得。伦理声明：本项目已获得广州市妇女儿童医疗中心医学伦理委员会批准。按照“赫尔辛基宣言”中所述的“涉及人体研究的国际伦理准则”进行实验。项目研究的知情同意书将从患者或患者的法定监护人获得。受试者的入选标准：本研究的受试者包括两类患者：一是在参与机构的儿童肝胆外科由经验丰富的医生 诊断高度怀疑BA的患者中招募，患者有计划接受肝胆Kasai手术，纳入标准：①0～90天龄梗阻性黄疸患儿；②经术中胆道造影明确诊断BA；③监护人知情同意；④不伴随癫痫等神经系统疾病；⑤不伴有原因不明的贫血；⑥可以进行规律随访；二是诊断为胆道闭锁患者已完成Kasai手术术后进行规律随访的患者中招募，纳入标准：①1～12个月龄患儿，已明确诊断BA；②已接受Kasai手术治疗；③监护人知情同意；④不伴有原因不明的贫血；⑤不伴有癫痫等神经系统疾病。此次试验共募集疾病组BA患者152名，对照组胆总管囊肿患者63名。
排除标准：(1)合并全身炎症反应综合征或多系统畸形的患儿；(2)原发疾病诊断不明确的患儿；(3)父母拒绝参加研究或无法获得父母授权的患儿；(4)未进行葛西手术的患者；(5)不能及时随访的患者；(6)恶性贫血及疑有维生素B12缺乏的病人。
退出标准：研究者实施的退出：(1)受试者在试验过程中出现重要器官功能异常；(2)受试者在试验过程中出现严重不良药物过敏反应；(3)受试者在试验过程中依从性差；(4)受试者在试验过程中病情加重或出现严重不良反应需要停止试验用药或采取其他治疗方法。受试者实施的退出：(1)受试者监护人感觉疗效不佳主动退出试验；(2)受试者监护人希望采取其他治疗方法主动退出试验；(3)受试者监护人无任何理由主动退出试验。
血清标本取得：
在患者监护人知情同意下，在入院、术中以及术后分别获取患者外周血0.5～1mL，用紫头EDTA抗凝管收集后放置冰盒中转运至实验室处理。实验室收到标本后首先留取0.3mL全血直接冻存-80℃冰箱，剩余的血液离心(1800rpm离心5min)后收集血清，并冻存。剩余的血细胞加入外周血淋巴细胞分离液(2mL)或者直接加入红细胞裂解液(4mL)进行分离或者裂解得到无红细胞的单个核细胞(PBMC)，进行细胞计数，冻存或者进行下一步的流式细胞学染色。
肝脏标本的取得：
在患者监护人知情同意下，对术中获取的肝脏标本，送病理固定切片后剩余部分放入组织保存液，在冰盒内转运至实验室。实验室接收到标本以后，将标本分成两份处理，一部分在去除组织表面血液后直接冻存在-80℃冰箱；剩余部分进行研磨，离心(1800rpm离心5min)，获取并冻存上清，剩余的沉淀在去除肝细胞后进行流式细胞学的染色。
BA患儿肠道标本的取得：在患者监护人知情同意下，取做Kasai手术造瘘口的肠管，放入组织保存液，在冰盒内转运至实验室。实验室接收标本后，将标本分成三份，一份固定送石蜡包埋病理切片；一份液氮速冻，转至-80℃冰箱冻存；第三部分剪碎放入含10mL胶原酶消化液(胶原酶II，DNA酶抑制剂，胎牛血清，RPMI1640培养基)的摇瓶中37℃摇床进行消化，进行过滤和红细胞裂解，最后重悬在PBS中，进行流式细胞学染色。对照疾病患儿胆总管囊肿肠道标本的取得则同样在患者监护人知情同意下，做手术时取样，并经历相同的标本处理步骤得到标本。
胚胎肠道标本的取得：在签署主动中止妊娠同意书以及生物医学研究的知情同意书后，妇产科协助获取胚胎标本。在体外剥离胎盘，剪断脐带，打开腹腔，取出肠道，进行4％多聚甲醛冰上固定2小时，后转入30％蔗糖固定过夜，第二天用OCT包埋固定，进行冰冻切片。
实施例2.血清叶酸检测
1.血清叶酸(Serum Folic Acid)检测：
取实施例1中的血清标本进行叶酸含量的检测，其中BA患者55例，以及年龄小于9个月的同龄对照疾病(胆总管囊肿)18例，按照叶酸检测试剂盒(Novus，货号：NBP2-59966-1Kit)的方法进行检测：在酶标包被板上设标准品孔10孔，设空白孔(空白对照孔不加样品及酶标试剂，其余各步操作相同)、待测样品孔。在酶标包被板上待测样品孔中先加样品稀释液40μL，然后再加待测样品10μL(样品最终稀释度为5倍)。用 封板膜封板后置37℃温育30分钟。弃去液体，甩干，每孔加满洗涤液，静置30秒后弃去，如此重复5次，拍干。每孔加入酶标试剂50μL，空白孔除外。温育30分钟，洗涤液洗涤。每孔先加入显色剂A50μL，再加入显色剂B50μL，轻轻震荡混匀，37℃避光显色15分钟。每孔加终止液50μL，终止反应(此时蓝色立转黄色)。以空白孔调零，450nm波长依序测量各孔的吸光度(OD值)。测定应在加终止液后15分钟以内进行。此外，采用ROC曲线(接收者操作特征，receiver operating characteristic)、根据AUC(曲线下面积)来验证BA特异性临床指标的预测性能。
结果如下：图1中A所示：通过对比胆道闭锁(BA)和同龄对照患者胆总管囊肿(choledochal cyst,CC)，发现在BA患者中叶酸的血清水平显著降低，P＜0.05，表明BA患者存在叶酸缺乏；图1中B：进一步对上述预测结果进行ROC曲线验证，结果显示叶酸能够非常有效地区分BA受试者与CC病例(AUC＝0.745)，而以同样的手段，将叶酸用于区分BA受试者与健康人群患者发现，其AUC>0.75，表明叶酸可以作为BA的诊断或辅助诊断的标志物之一。
实施例3.叶酸对胆道闭锁小鼠模型的预防和治疗效果
1.胆道闭锁动物模型的建立：
(1)动物：成年BALB/c孕鼠，无特定病原体级(SPF级)，购自广东省医学实验动物中心。饲养在广州医科大学实验动物中心SPF级环境。待孕鼠产下新生小鼠(每只孕鼠平均产8只新生鼠)，平均体重1.5g，根据实验分组随机选取新生小鼠进行实验。本实验动物处置方法符合动物伦理学标准。
(2)造模方法：将新生的BALB/c小鼠，于出生24小时内腹腔注射猴MMU18006轮状病毒(下称“RRV”)20μL(滴度1.0×10 6PFU)，建立BA小鼠动物模型。
(3)观察小鼠的生存状况：包括小鼠生存率、生长体重、皮肤黄疸和肝功能的变化等。
2.叶酸的效果实验
在出生24小时内感染恒河猴轮状病毒(RRV)建立BA模型，在已建立的急性胆道闭锁小鼠模型上，根据实验要求进行配对实验，将BA模型分为不同实验组别：1)对照小鼠组(Control，腹腔给与同等体积的PBS，n＝9)；2)RRV诱导BA小鼠模型组(RRV，具体方法同步骤1，n＝7)；3)叶酸补充组(RRV+FA，n＝11)，在RRV诱导BA小鼠模型基础上给与叶酸治疗，其中，叶酸给药剂量为6.25mg/kg，腹腔注射，病毒感染后16小时给药，每天给药一次，实验在第13天终止(具体步骤如图2中A)。在实验的整个过程中观察和记录上述各组小鼠的体重、肝胆表观、黄疸特征及生存率的同时，流式细胞学检测肝脏免疫细胞亚型，ELISA检测叶酸的水平。
结果如下：
图2中B为小鼠的体重随着时间的变化图，从图2中B中可见，从造模的第11天起，RRV诱导BA小鼠模型组与对照小鼠组、叶酸补充组小鼠的体重开始出现显著差异(p＜0.01)，尤其到第13天时，RRV诱导BA小鼠模型组分别与对照小鼠组、叶酸补充组之间的体重显著差异P值达到0.001，而对照小鼠组和叶酸补充组之间的体重则没有显著差异(p＞0.05)。由此可见，叶酸的补充显著改善了RRV感染所致的BA模型小鼠体重减轻。
图2中C为小鼠的黄疸率随着时间的变化图，从图2中C中可见，在实验终止的第13天时，对照小鼠组的黄疸率为0％、RRV诱导BA小鼠模型组的黄疸率为80％、叶酸补充组小鼠的黄疸率为30％，且RRV诱导BA小鼠模型组的黄疸率与叶酸补充组小鼠的黄疸率出现极显著性差异(p＝0.0003)。可见，BA模型小鼠造模成功，而叶酸补充组显著改善了BA模型小鼠所降低的黄疸率，黄疸率由RRV组80％降至30％，表现出显著治疗的效果。
图2中D为小鼠肝脏中坏死灶或淋巴细胞的情况表示图，从图2中D中可见，在实验终止的第13天时，RRV诱导BA小鼠模型组的坏死灶和淋巴细胞浸润情况严重，而叶酸补充组小鼠肝脏中坏死灶以及淋巴细胞 浸润减少，和对照小鼠的坏死灶和淋巴细胞浸润情况接近，较不明显。可见，叶酸在BA疾病中表现出显著的抗炎治疗效果。
图2中E为肝脏中CD4 +T细胞中的IFN-γ含量图，从图2中E中可见，在实验终止的第13天时，RRV诱导BA小鼠模型组分别与对照小鼠组、叶酸补充组之间的肝脏中CD4 +T细胞中的IFN-γ含量呈现显著差异(p＜0.01)，而对照小鼠组和叶酸补充组之间的CD4 +T细胞中的IFN-γ含量则没有显著差异(p＞0.05)。可见相对于RRV诱导BA小鼠模型组，叶酸补充组中的细胞毒性细胞因子IFN-γ在肝脏的表达显著减少，叶酸表现出显著的BA疾病中的抗炎、抗病毒治疗效果，提示补充叶酸抑制T细胞分泌毒性细胞因子IFN-γ而对BA肝脏起保护作用。
3.HE染色实验
肠道苏木精和伊红(H&E)染色(或简称HE染色)。HE染色的具体方法如下：取动物组织固定于4％多聚甲醛(PFA)固定24h，依次浸入50％、60％、70％、80％、90％和100％乙醇脱水，2次二甲苯透明，石蜡包埋，制4μm切片。60℃烤片后，石蜡切片依次经过2次二甲苯，每次15min脱蜡，依次经过2次100％和70％乙醇复水，蒸馏水浸洗2次。苏木素室温染色6min，流水洗去浮色。1％盐酸酒精分色3s，置于自来水中，流水返蓝5min。伊红染色3min，依次经过2次70％、100％乙醇脱水，经过2次二甲苯透明，中性树脂封片并镜检。
小鼠肠道的HE染色结果如图3、4所示。从图3中可见，在实验的第12天时，RRV诱导BA小鼠模型组出现显著的肠道上皮异常的空泡样变性，而叶酸补充组仅出现轻微的肠道上皮空泡样变性，接近空白对照组的表征；从图4中可见，相对于RRV诱导BA小鼠模型组，叶酸补充组BA小鼠肠道绒毛长度显著增长，组织结构明显更紧密，与空白对照接近；可见叶酸补充组的RRV感染诱导的肠道上皮异常的空泡样变性、组织结构损伤均得到显著修复。
综上所述，以上结果说明叶酸通过抑制毒性细胞因子的表达以及修复肠道屏障对BA小鼠起到显著的治疗作用。再者，从图2中B、C可见，RRV诱导胆道闭锁模型在7天左右出现明显症状，而本实验在造模第一天的同时已进行叶酸给药，所以以上结果不仅证明了叶酸对于BA疾病的治疗效果，还证明了叶酸对BA的预防效果。同时，发明人在后续研究中，通过预先给与叶酸和设置空白对照，再进行RRV病毒感染从而BA造模的方式，进一步证明了叶酸对于BA的预防效果。
实施例4 S100a8、S100a9、Nox2、IFN-γ用于胆道闭锁的诊断
1、qPCR检测炎症因子的表达
对实施例3中的对照小鼠组；RRV诱导BA小鼠模型组；叶酸补充组三组的小鼠肠道提取RNA，RNA采用Trizol一步法提取。cDNA第一链的合成采用Promega反转录试剂盒进行。根据跨内含子原则进行引物设计以避免基因组污染干扰。引物序列如下：
S100a8:F:AAATCACCATGCCCTCTACAAG(SEQ ID NO.1)；R:CCCACTTTTATCACCATCGCAA(SEQ ID NO.2)；
S100a9:F:ATACTCTAGGAAGGAAGGACACC(SEQ ID NO.3)；R:TCCATGATGTCATTTATGAGGGC(SEQ ID NO.4)；
IFN-γ:F:CCGAACTGTACGGACATCACA(SEQ ID NO.5)；R:TCATAGTGTTGAAATGGCTCCAG(SEQ ID NO.6)；
Nox2:F:TTCCCCCAATTCAATGTCTGG(SEQ ID NO.7)；R:TCCACCTGACACGACTTGAGA(SEQ ID NO.8)。
采用Eva Green荧光染料法进行实时定量PCR扩增的检测。靶基因相对表达量采用2-ΔCT进行计算。
结果如下：
在对肠道组织的qPCR检测发现，如图5中的基因S100a8表达情况图所示，在RRV诱导BA小鼠模型组中，基因S100a8异常高表达，在叶酸补充组中仅稍高于空白对照组，基因S100a8在RRV诱导BA小鼠模型组中的表达显著区别于叶酸补充组和空白对照组(p＜0.05)，而在叶酸补充组和空白对照组中，基因S100a8的表达差异不显著。由此可见，S100a8基因(以及应理解为相应的其蛋白)可以作为胆道闭锁疾病的诊断或辅助诊断的有效标志物之一，且当检测到S100a8基因或其蛋白相对于未患有胆道闭锁疾病的同年龄段受试者相应指标的水平高表达时，则可认为样本所来自个体患有胆道闭锁或患有胆道闭锁的可能性较高。
如图5中的基因S100a9表达情况图所示，在RRV诱导BA小鼠模型组中，基因S100a9异常高表达，显著区别于空白对照组(p＜0.05)；在叶酸补充组中其表达虽然高于空白对照组，但对比在RRV诱导BA小鼠模型组中，仍然取得了有效降低基因S100a9表达的效果。由此可见，S100a9基因(以及应理解为相应的其蛋白)可以作为胆道闭锁疾病的诊断或辅助诊断的有效标志物之一，且当检测到S100a9基因或其蛋白相对于未患有胆道闭锁疾病的同年龄段受试者相应指标的水平高表达时，则可认为样本所来自个体患有胆道闭锁或患有胆道闭锁的可能性较高。
如图5中的基因IFN-γ表达情况图所示，在RRV诱导BA小鼠模型组中，基因IFN-γ异常高表达，在叶酸补充组中仅稍高于空白对照组，基因IFN-γ在RRV诱导BA小鼠模型组中的表达显著区别于叶酸补充组和空白对照组(p＜0.05)，而在叶酸补充组和空白对照组中，基因IFN-γ的表达差异不显著。由此可见，IFN-γ基因(以及应理解为相应的其蛋白)可以作为胆道闭锁疾病的诊断或辅助诊断的有效标志物之一，且当检测到IFN-γ基因或其蛋白相对于未患有胆道闭锁疾病的同年龄段受试者相应指标的水平高表达时，则可认为样本所来自个体患有胆道闭锁或患有胆道闭锁的可能性较高。
如图5中的基因Nox2表达情况图所示，在RRV诱导BA小鼠模型组中，基因Nox2异常低表达，显著区别于空白对照组(p＜0.05)；在叶酸补充组中其表达虽然低于空白对照组，但对比在RRV诱导BA小鼠模型组中，仍然取得了有效提高基因Nox2表达的效果。由此可见，Nox2基因(以及应理解为相应的其蛋白)可以作为胆道闭锁疾病的诊断或辅助诊断的有效标志物之一，且当检测到Nox2基因或其蛋白相对于未患有胆道闭锁疾病的同年龄段受试者相应指标的水平低表达时，则可认为样本所来自个体患有胆道闭锁或患有胆道闭锁的可能性较高。
综上所述，BA小鼠模型中S100a8、S100a9、IFN-γ、Nox2的表达量显著区别于空白对照组，因此，S100a8、S100a9、IFN-γ和/或Nox2可以作为胆道闭锁疾病的诊断或辅助诊断的有效标志物之一。并且，进一步地，通过叶酸的补充，可以降低BA模型小鼠肠道炎症，从而达到防治胆道闭锁引起的肠道疾病的目的。
在实施例2的基础上，发明人通过实验发现，采用血清中的叶酸，联合肠道组织中的S100a8、S100a9、IFN-γ、Nox2中的一种或多种，可以更加准确地用于胆道闭锁的诊断或辅助诊断，表现为更高的准确性、特异性，和更高的AUC值。
实施例5.亚叶酸钙对胆道闭锁小鼠模型的预防和治疗效果
1.胆道闭锁动物模型的建立：
(1)动物：成年BALB/c孕鼠，无特定病原体级(SPF级)，购自广东省医学实验动物中心。饲养在广州医科大学实验动物中心SPF级环境。待孕鼠产下新生小鼠(每只孕鼠平均产8只新生鼠)，平均体重1.5g，根据实验分组随机选取新生小鼠进行实验。本实验动物处置方法符合动物伦理学标准。
(2)造模方法：将新生的BALB/c小鼠，于出生24小时内腹腔注射猴MMU18006轮状病毒(下称“RRV”)20μL(滴度1.0×10 6PFU)，建立BA小鼠动物模型。
(3)观察小鼠的生存状况：包括小鼠黄疸发生率、生长体重、肝肠表现等。
2.亚叶酸钙的效果实验
在出生24小时内感染恒河猴轮状病毒(RRV)建立BA模型，在已建立的急性胆道闭锁小鼠模型上，根 据实验要求进行配对实验，将BA模型分为不同实验组别：1)对照小鼠组(Control，腹腔给与同等体积的PBS)；2)RRV诱导BA小鼠模型组(RRV，具体方法同步骤1)；3)亚叶酸钙补充组(RRV+CF)，在RRV诱导BA小鼠模型基础上给与亚叶酸钙治疗，其中，亚叶酸钙给药剂量为5mg/kg，腹腔注射，病毒感染后16小时给药，每天给药一次，实验在第12天终止。在实验的整个过程中观察和记录上述各组小鼠的体重、肝肠表现以及黄疸发生率的同时，检测肝脏中INFβ、ROS水平，小肠中Dhfr、Mthfr、Nrf2、Slc40a1，流式细胞学检测肝脏免疫细胞亚型，结果如7所示：
从造模的第10天起，RRV诱导BA小鼠模型组与对照小鼠组、亚叶酸钙补充组小鼠的体重开始出现显著差异(p＜0.01)，而对照小鼠组和亚叶酸钙补充组之间的体重则没有显著差异(p＞0.05)(图7中A)；
在实验终止的第12天时，亚叶酸钙补充组显著降低了BA模型小鼠的肝脏门管区周围淋巴细胞浸润；亚叶酸钙补充组显著增加了BA模型小鼠的肝小叶结构的完整性；亚叶酸钙补充组使BA模型小鼠的肠上皮细胞的气球样变性得到恢复(图7中B)；
在实验终止的第12天时，对照小鼠组的黄疸率为0％、RRV诱导BA小鼠模型组的黄疸率为100％、亚叶酸钙补充组小鼠的黄疸率为不到70％，且RRV诱导BA小鼠模型组的黄疸率与亚叶酸钙补充组小鼠的黄疸率出现显著性差异(p＜0.05)，可见，BA模型小鼠造模成功，而亚叶酸钙补充组显著降低了BA模型小鼠的黄疸率，黄疸率由RRV组100％降至不到70％，表现出显著治疗的效果(图7中C)；
在实验终止的第12天时，RRV诱导BA小鼠模型组与对照小鼠组、亚叶酸钙补充组小鼠的肝脏中INFβ存在显著差异(p＜0.01)，RRV诱导BA小鼠模型组与对照小鼠组、亚叶酸钙补充组小鼠的肝脏中ROS存在显著差异(p＜0.05)；亚叶酸钙补充组显著降低了BA模型小鼠的肝脏中INFβ、ROS的含量(图7中D)；
在实验终止的第12天时，RRV诱导BA小鼠模型组与对照小鼠组、亚叶酸钙补充组小鼠的小肠中Dhfr、Mthfr、Nrf2、Slc40a1的表达量存在显著差异(p＜0.05)，亚叶酸钙补充组显著提高了BA模型小鼠的小肠中Dhfr、Mthfr、Nrf2、Slc40a1的表达量(图7中E)；
在实验终止的第12天时，RRV诱导BA小鼠模型组分别与对照小鼠组、叶酸补充组之间的肝脏中CD4 +T细胞、CD8 +T细胞中的IFN-γ含量呈现显著差异(p＜0.001)，亚叶酸钙补充组显著降低了BA模型小鼠的肝脏中CD4 +T细胞、CD8 +T细胞中的IFN-γ含量；RRV诱导BA小鼠模型组分别与对照小鼠组、叶酸补充组之间的肝脏中浆细胞中IgG1含量呈现显著差异(p＜0.001)，亚叶酸钙补充组显著降低了BA模型小鼠的肝脏中浆细胞中IgG1含量(图7中F)；
因此，补充亚叶酸钙(CF)可降低肝脏中的INFβ、ROS的含量，降低黄疸发生率并改善新生儿生长；提高小肠中Dhfr、Mthfr、Nrf2、Slc40a1的表达量，从而使叶酸代谢、氧化应激和铁稳态得到恢复；降低肝脏中CD4 +T细胞、CD8 +T细胞中的IFN-γ含量、浆细胞中IgG1含量；降低肝脏门管区周围淋巴细胞浸润；增加肝小叶结构的完整性；使肠上皮细胞的气球样变性得到恢复，达到治疗胆道闭锁的效果。
实施例6.叶酸对胆道闭锁婴儿胆管炎的发病率的影响以及治疗机制的研究
在一项临床研究中，19名BA受试者以0.4mg/天的剂量口服叶酸，持续6个月；同时，招募了一组年龄(在Kasai手术时)、性别、肝酶、胆汁酸和胆红素状态等临床特征匹配的BA受试者(n＝38)作为对照来比较叶酸治疗效果(如图8中A、B所示)。
结果发现，细菌性胆管炎的总发病率从未治疗的74％(28/38)下降到叶酸治疗患者的21％(4/19)(图8中C、图9中A)。此外，在3个月的随访中观察到叶酸治疗患者的总胆红素浓度和直接胆红素浓度显著降低(图9中B、C)。
发明人还发现，在胆管炎患者中，服用叶酸和未服用叶酸，也表现出了显著的总胆红素浓度和直接胆红素浓度的差异，叶酸治疗患者的总胆红素浓度和直接胆红素浓度显著降低，从而表明了叶酸对于胆管炎治疗的效果。
进一步地，发明人通过后续研发发现，在胆管炎患者中，联合服用叶酸和维生素B12，与未服用相比，也表现出了显著的总胆红素浓度和直接胆红素浓度降低的效果，从而表明了叶酸和维生素B对于胆管炎的联合治疗效果。
综上所述，一方面，胆管炎是胆道闭锁发生发展中的重要病理特征，另一方面，胆管炎是胆道闭锁术后的最常见并发症，本发明发现的叶酸及其盐，不仅可以用于预防胆道闭锁并发症胆管炎的发生，此外，也可应用于胆管炎疾病的治疗中。
为了进一步研究叶酸的治疗机制，使用BD Rhapsody平台生成了一个额外的scRNA转录组数据集(图10中A)，并将分析重点放在肠上皮细胞上(图10中B)。治疗后，调节叶酸吸收(FOLH1)、铁输出(FTH1、NCOA4、SLC40A1)和抑制脂质过氧化(NFE2L2/NRF2、SELENOP、GPX4)的基因显著上调，相反，对IFN-I的反应(STAT1、IFITM2、IFITM3)在接受叶酸治疗的患者中显着降低(图10中C、图11中A、B)。叶酸处理也显著增加了循环中四氢生物蝶呤(BH4)的浓度，但降低了肝脏中同型半胱氨酸的浓度(图11中C)。肠上皮细胞中SLC40A1表达的增加证明了全身铁超载和腔内铁缺乏的纠正。在术前接受叶酸治疗的患者中，血浆中铁水平下降，但10例粪便铁水平升高(图12中A)。叶酸治疗的其他治疗益处包括抑制小肠嗜酸性粒细胞浸润，抑制IgG-Ro/SSA自身抗体的产生，增加IgM/IgG4比值(图12中B)，这可能预测BA的肝脏健康和小肠和肝脏骨髓细胞中IFN-I信号通路的抑制。
因此，补充叶酸可以降低胆道闭锁患者的总胆红素浓度和直接胆红素浓度，达到预防和/或治疗胆管炎的效果。同时，补充叶酸还可以达到纠正全身铁超载和管腔铁缺乏、抑制小肠嗜酸性粒细胞浸润、抑制IgG-Ro/SSA自身抗体的产生，增加IgM/IgG4比值，同时，延长自身免疫期和改善肝功能。
实施例7亚叶酸钙对病毒细菌感染的预防和治疗效果
1.骨髓来源巨噬细胞的分化
1)小鼠取腿骨
小鼠麻醉后脱臼处死，将小鼠放置在盛有足量75％乙醇的烧杯中浸泡消毒5min，将浸泡好的动物用纸吸去多余的酒精；用剪刀在小鼠背部剪开一小口，用手直接撕开皮肤至小鼠小腿关节处，去除小鼠足关节以及皮肤；用剪刀沿着小鼠大腿根部大转子将后肢拆下来，去掉肌肉组织后放置在含有75％乙醇的培养皿内浸泡5min，更换新的75％乙醇的培养皿移入超净台中。
2)骨髓巨噬细胞(BMDM)提取和诱导
将乙醇浸泡的腿骨移入冷的PBS浸泡，洗去胫骨、股骨表面的乙醇，此过程可重复3次；将清洗好的股骨、胫骨分开，并用剪刀分别将股骨、胫骨两端剪断，使用1mL注射器吸取冷的诱导培养基将骨髓从股骨、胫骨中吹出，反复吹洗3次，直至腿骨内看不到明显的红色为止；用5mL移液枪将含有骨髓细胞的培养基反复吹打，使细胞团块分散，然后使用70μm细胞滤器将细胞过筛，转移至15mL离心管内，1500rpm/min离心5min，弃上清，加入红细胞裂解液重悬静置5min后1500rpm/min离心5min，弃上清用冷的配置好的骨髓巨噬细胞诱导培养基重悬，铺板；细胞培养期间不更换培养基，培养第三天后更换一半骨髓巨噬细胞诱导培养基，第五天更换全部培养基，第七天即可用于后续实验。
2.进行病毒模拟抗原、病毒或者细菌来源的脂多糖刺激
吸去培养皿中的培养基，用PBS或者无血清培养基清洗一次；更换无血清培养基；准备转染制备液，用灭菌后的EP管制备；以六孔板为例，A液：用200μl Opti-MEM稀释poly(I:C)(poly(I:C)(聚肌苷酸胞苷酸)是一种合成的双链RNA(dsRNA)类似物，模拟双链RNA病毒感染(刺激浓度5ug/ml))或者poly dAdT(Poly(dA:dT)(聚脱氧腺苷酸)，是一种双链交替共聚物DNA模型，模拟DNA病毒感染(5ug/ml))；B液:用200μl Opti-MEM稀释10μl lipo2000，分别将A液、B液轻轻混匀，静置5min，吸取B液加入至A液中，轻轻混匀，室温静置20min；加入转染试剂或仙台病毒(仙台病毒(Sev)是反义RNA病毒，模拟反义RNA 病毒感染)或LPS((Sigma，刺激浓度：10ng/ml)来源于细菌的脂多糖，模拟细胞感染)到每个孔的培养基中(Blank组不加任何物质)，同时，实验组给予亚叶酸钙(Sigma，刺激浓度10ug/ml)刺激(对照组则加入等量溶剂)，培养到16～18小时，收集细胞或者培养上清进行I-IFN通路激活的检测。
3.ELISA方法检测IFN-β
白色平底
Figure PCTCN2022130161-appb-000001
96孔板(Thermo Fisher)在室温下用50μl mIFN-β捕获抗体包被过夜。封闭后，加入50ul培养液、标准品和30ng/ml lucia-conjugated检测抗体，37℃孵育2h。洗涤后，加入50ul重构的QUANTI-Luc TM Plus，立即通过化学发光检测。酶标仪预先设置，读取时间设置为0.5s。
结果如图6所示：细菌、反义RNA病毒、DNA病毒等可引起的体内过度激活的I-IFN通路，而通过补充叶酸或其衍生物(亚叶酸钙)可以抑制上述细菌和/或病毒感染引起的I-IFN的过度激活，即通过补充叶酸或其衍生物(亚叶酸钙)可以预防和/或治疗细菌和/或病毒感染，从而达到预防和/或治疗新生儿感染疾病的效果。
在此基础上，发明人进一步通过实验研究发现，叶酸或其衍生物与活性成分维生素B、维生素C和牛磺酸中的至少一种联合，特别是与维生素B6、维生素B12等联合，表现出了更加优异的针对上述细菌、病毒感染的抑制效果。
实施例8叶酸与维生素B联用对胆道闭锁小鼠模型的预防和治疗效果
1.胆道闭锁动物模型的建立：
(1)动物：成年BALB/c孕鼠，无特定病原体级(SPF级)，购自广东省医学实验动物中心。饲养在广州医科大学实验动物中心SPF级环境。待孕鼠产下新生小鼠(每只孕鼠平均产8只新生鼠)，平均体重1.5g，根据实验分组随机选取新生小鼠进行实验。本实验动物处置方法符合动物伦理学标准。
(2)造模方法：将新生的BALB/c小鼠，于出生24小时内腹腔注射猴MMU18006轮状病毒(下称“RRV”)20μL(滴度1.0×10 6PFU)，建立BA小鼠动物模型。
(3)观察小鼠的生存状况：小鼠黄疸发生率、生长体重。
2.效果实验
在出生24小时内感染恒河猴轮状病毒(RRV)建立BA模型，在已建立的急性胆道闭锁小鼠模型上，根据实验要求进行配对实验，将BA模型分为不同实验组别：1)对照小鼠组(Control，腹腔给与同等体积的PBS)；2)RRV诱导BA小鼠模型组(RRV，具体方法同步骤1)；3)叶酸和维生素B6补充组(RRV+CF+B6)，在RRV诱导BA小鼠模型基础上给与叶酸和维生素B6联合治疗，其中，叶酸给药剂量为3.5mg/kg，维生素B6给药剂量为2.5mg/kg，混合后腹腔注射，病毒感染后16小时给药，每天给药一次，实验在第12天终止。在实验的整个过程中观察和记录上述各组小鼠的体重、以及黄疸发生率。
从造模的第8天起，RRV诱导BA小鼠模型组与对照小鼠组、叶酸和维生素B6补充组小鼠的体重开始出现显著差异(p＜0.05)，而对照小鼠组和叶酸和维生素B6补充组之间的体重则没有显著差异(p＞0.05)；
在实验终止的第12天时，对照小鼠组的黄疸率为0％、RRV诱导BA小鼠模型组的黄疸率为100％、叶酸和维生素B6补充组小鼠的黄疸率为45％，且RRV诱导BA小鼠模型组的黄疸率与叶酸和维生素B6补充组小鼠的黄疸率出现显著性差异(p＜0.05)，可见，BA模型小鼠造模成功，而叶酸和维生素B6补充组显著降低了BA模型小鼠的黄疸率，黄疸率由RRV组100％降至45％，表现出显著治疗的效果。
在此基础上，发明人进一步通过实验研究证明，通过预先联合采用叶酸和维生素B6、和设置空白对照，再进行RRV病毒感染从而BA造模的方式，进一步证明了叶酸和维生素B6对于BA的预防效果。
实施例9叶酸降低胆道闭锁和胆总管囊肿患者食物过敏的发生
1、胆道闭锁患者术后食物过敏的发生
该调查由广州市妇女儿童医疗中心(GWCMC)小儿外科联合儿科研究所进行，使用电子表格获取信息。 样本共包含249个有效病例，其中154个来自BA患者，95个来自CC患者。参与者是通过在线小程序(兼容计算机和手机)招募的。参与者应邀完成了在线调查。结构化问卷的主要内容包括孕期和产后妈妈的营养摄入、产后营养与感染、术后并发症及婴儿过敏源。该调查已获得GWCMC医学伦理委员会的数据收集和分析批准。
调查结果如图13中的A所示，胆道闭锁中约有15.12％的患者在胆道闭锁术后出现食物过敏的现象。
2、叶酸抑制胆道闭锁患者术后食物过敏的发生
对胆道闭锁或疑似胆道闭锁的患者使用叶酸治疗7天进行手术治疗，取术中的肠道标本进行免疫荧光的染色，如图13中的B所示，发现肠道中参与食物过敏的嗜酸性粒细胞数量显著减少，这表明，叶酸对于胆道闭锁患者的食物过敏的发生具有预防和抑制作用。
3、叶酸抑制胆囊肿患者术后食物过敏的发生
如图13的A所示，胆总管囊肿术后的食物过敏发生率虽然比胆道闭锁术后的食物过敏发生率显著低，但是仍然有4.29％的患者在胆总管囊肿术后发现食物过敏的现象。食物过敏可能会导致胃肠道、皮肤、呼吸道等部位症状，目前的抗过敏药物的副作用大，而且治疗周期较长，有进一步加剧其副作用。
对于胆总管囊肿患者在术前使用叶酸治疗7天进行手术治疗，取术中的肠道标本进行免疫荧光的染色，同样发现在肠道中参与食物过敏的嗜酸性粒细胞数量显著减少，这表明，叶酸对于胆总管囊肿患者的食物过敏的发生也具有预防和抑制作用。
实施例10叶酸用于抑制坏死性小肠结肠炎
1、诱导NEC小鼠模型
在7-10日龄小鼠中诱导实验性NEC，对SPF级七日龄C57BL/6小鼠灌胃鼠代乳，然后将其置于90％N 2+5％CO 2+5％O 2，4℃环境中10min，随后取出。每日三次灌胃+造模，每次间隔8h，持续三天。
2、叶酸用于NEC小鼠
将NEC模型小鼠进行分组：(1)正常小鼠组，正常母乳喂养小鼠；(2)NEC模型小鼠组；(3)叶酸+NCE模型小鼠组，在NEC模型小鼠基础上给与叶酸，其中，叶酸给药剂量为6mg/kg，腹腔注射，16小时给药，每天给药一次，实验在第6天终止。终止后处死小鼠，取肠道组织观察，并且提取肠段组织的RNA，进行qPCR，检测肠段组织中IFN-γ、IL-1β的表达量。
经过观察和实验发现，与正常小鼠组相比，NEC模型小鼠组的肠段表现出严重粘膜损伤及斑片状溃疡，而叶酸给药组的小鼠肠段的粘膜坏死及溃疡情况都有不同程度的可观察到的减轻，而肠段基因检测发现，与NEC模型小鼠组相比，叶酸给药组的炎症因子都受到了明显的抑制。这表明，叶酸对于NEC具有显著的治疗作用。而在进一步的研究中，在预先给与叶酸的NEC小鼠中也观察到了相似的情形，表现为粘膜损伤的减轻和炎症因子的抑制。
因此，叶酸对于NEC表现出明显的预防和治疗作用。
实施例11叶酸用于新生儿巨细胞病毒感染(HCMV)的预防和治疗
收集约20例临床诊断或者高度怀疑为HCMV感染的尿液，检测指标为尿液HCMV拷贝数，设置对照组(其他一致，仅未服用叶酸)和服用叶酸组，服用剂量为0.4mg/天，每天一次，坚持服用10天，每天检测尿液HCMV拷贝数。与未服用叶酸组相比，服用叶酸组的尿液HCMV拷贝数在更短的时间内得到了控制，平均天数缩短1-2天，在3、6、10天采集患儿血液进行炎症因子检测，发现，服服用叶酸组的TNF-α、IL-1β炎症因子水平更低，特别是在第6天，其差异显著。
新生儿的巨细胞病毒感染，影响婴儿的发育并且可能导致肝脏、中枢神经系统等各处损伤，特别是，因此，本发明中通过证明叶酸可以抑制巨细胞病毒感染，体现了叶酸用于新生儿巨细胞病毒感染的预防和治疗前景。
综上所述，本发明证明了，BA中血清叶酸显著降低(图1中A)、叶酸水平能够有效地区分BA与非BA受试者(图1中B)，因此可以通过孕期补充叶酸起到预防胆道闭锁的效果；叶酸补充可以增加BA体重(图2中B)、可以降低黄疸率(图2中C)，同时可以抗炎、减轻肝脏组织损伤以及纤维化程度(图2中D)、降低IFN-γ水平(图2中E)、修复肠道损伤(图3、4)；肠道组织炎症因子(S100a8、S100a9、IFN-γ)表达降低同时抑炎基因(Nox2)表达升高(图5)，S100a8、S100a9、IFN-γ或Nox2水平能够有效地区分BA与非BA受试者；并且，本发明还验证了S100a8、S100a9、IFN-γ、Nox2、叶酸中的至少两种组成的标志物组合物能够有效地区分BA与非BA受试者。因此，叶酸或其盐可有效防治胆道闭锁、胆管炎、病理性黄疸，且本发明证明了补充叶酸能减轻病毒感染性胆道闭锁小鼠模型中的黄疸率、改善小鼠肝脏/肠道功能、抑制肝脏细胞毒性细胞因子IFN-γ的表达，还可以降低胆道闭锁患者的总胆红素浓度和直接胆红素浓度(图9中B、C)，达到预防和/或治疗胆管炎的效果(图8中C、图9中A)。同时，补充叶酸还可以达到纠正全身铁超载和管腔铁缺乏、抑制小肠嗜酸性粒细胞浸润、抑制IgG-Ro/SSA自身抗体的产生，增加IgM/IgG4比值(图12中B)，同时，延长自身免疫期和肝脏存活率；本发明还证明了：补充亚叶酸钙(CF)可降低肝脏中的INFβ、ROS的含量(图7中D)，降低黄疸发生率并改善新生儿生长(图7中A、C)；提高小肠中Dhfr、Mthfr、Nrf2、Slc40a1的表达量(图7中E)，从而使叶酸代谢、氧化应激和铁稳态得到恢复；降低肝脏中CD4 +T细胞、CD8 +T细胞中的IFN-γ含量、浆细胞中IgG1含量(图7中F)；降低肝脏门管区周围淋巴细胞浸润；增加肝小叶结构的完整性；使肠上皮细胞的气球样变性得到恢复(图7中B)，达到治疗胆道闭锁的效果；通过补充叶酸或其衍生物(亚叶酸钙)可以预防和/或治疗细菌和/或病毒感染，从而达到预防和/或治疗新生儿感染疾病的效果，并且对于坏死性小肠结肠炎也表现出明显的抑制效果；因此，本发明还成功地提供了一种能同时作用于肝、肠等消化道的安全活性成分——叶酸，从而，本发明除了可防治胆道闭锁外，还可通过其作用靶点的多样性，达到对肝纤维化病、胆管炎、黄疸、肝肠循环障碍、病毒感染、炎症性肠病、肠道菌群失调等疾病的防治作用。最后，本发明还提供了叶酸、S100a8、S100a9、Nox2、IFN-γ中的一种或多种作为胆道闭锁的诊断或辅助诊断标志物的用途。因此，本发明不仅发现叶酸可用于诊断、防治胆道闭锁等疾病，还通过对机理的探究扩展了其应用前景。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (21)

  1. 叶酸或其衍生物在制备预防和/或治疗遗传性、感染性或过敏性疾病的药物中的应用，其特征在于，所述遗传性、感染性或过敏性疾病包括胆道闭锁、胆管炎、黄疸、新生儿感染性疾病、坏死性小肠结肠炎、细菌感染性疾病、病毒感染性疾病、食物过敏。
  2. 根据权利要求1所述的应用，其特征在于，所述胆道闭锁包括病毒引起的胆道闭锁、或胆汁淤积引起的胆道闭锁；
    优选的，所述病毒引起的胆道闭锁还包括主要由巨细胞病毒引起的胆道闭锁、或主要由轮状病毒引起的胆道闭锁；
    其中，所述胆道闭锁还表现为胆道闭锁引起的食物过敏、胆道闭锁引起的黄疸、胆道闭锁引起的胆管炎、胆道闭锁引起的肝脏疾病、胆道闭锁引起的肠道性疾病；
    优选的，所述胆道闭锁引起的肝脏疾病包括胆道闭锁引起的肝脏功能损伤、和/或胆道闭锁引起的肝脏炎症性疾病；
    所述胆道闭锁引起的肠道性疾病包括胆道闭锁引起的肠道性炎症性疾病。
  3. 根据权利要求1所述的应用，其特征在于，所述黄疸包括病理性黄疸或婴儿期黄疸；
    优选的，所述病理性黄疸包括病毒引起的病理性黄疸、或婴儿期病理性黄疸；
    优选的，所述病理性黄疸还包括病毒引起的婴儿期病理性黄疸；
    优选的，所述病毒引起的病理性黄疸包括巨细胞病毒性黄疸。
  4. 根据权利要求1所述的应用，其特征在于，所述胆管炎包括细菌性胆管炎、病毒性胆管炎、胆道闭锁引起的胆管炎、胆道闭锁术后并发的胆管炎；
    优选的，所述细菌性胆管炎还包括胆道闭锁引起的细菌性胆管炎、胆道闭锁术后并发的细菌性胆管炎。
  5. 根据权利要求1所述的应用，其特征在于，所述新生儿感染性疾病还包括新生儿细菌感染疾病和新生儿病毒感染疾病、新生儿支原体或者新生儿衣原体感染；
    优选的，所述新生儿病毒感染还包括RNA病毒和DNA病毒；
    进一步优选的，所述RNA病毒还包括反义RNA病毒、DNA病毒；
    其中，所述新生儿病毒感染还包括新生儿巨细胞病毒(CMV)感染、新生儿轮状病毒感染、新生儿疱疹病毒感染；
    优选的，所述新生儿感染性疾病包括新生儿肝炎综合征、新生儿败血症、新生儿肺炎、新生儿感染导致的胃肠道疾病。
  6. 根据权利要求1所述的应用，其特征在于，所述细菌感染性疾病包括大肠杆菌、金黄色葡萄球菌或溶血性链球菌；所述病毒感染性疾病包括巨细胞病毒(CMV)感染、轮状病毒感染、疱疹病毒感染；其中，所述病毒性感染疾病还包括病毒性腹泻。
  7. 根据权利要求1所述的应用，其特征在于，所述食物过敏包括胆道闭锁引起的或者胆道闭锁伴随的食物过敏、胆总管囊肿引起的或胆总管囊肿伴随的食物过敏；或，所述食物过敏还表现为食物过敏导致的皮肤疾病、食物过敏导致的胃肠道疾病、食物过敏导致的呼吸道疾病；优选的，所述食物过敏导致的胃肠道疾病包括食物过敏导致的炎症性肠病。
  8. 根据权利要求1-7中任一项所述的应用，其特征在于，所述叶酸衍生物包括(a1)～(a2)中任一种：
    (a1)亚叶酸、二氢叶酸、四氢叶酸、5-甲基四氢叶酸、5,10-亚甲基四氢叶酸、5,10-亚甲基四氢叶酸、5,10-甲酰亚氨基四氢叶酸、5-甲酰四氢叶酸、10-甲酰四氢叶酸和10-甲基四氢叶酸；
    (a2)为(a1)的药学上可接受的盐；
    优选的，所述药学上的盐包括钙、钠、锌、精氨酸、胆碱、乙酰胆碱、N-甲基氨基乙醇、2-氨基-2-甲基-丙醇、1,1-二甲基双胍、苯乙基双胍、二氨基胍、葡糖胺和二甲基氨基乙醇；
    进一步优选的，所述叶酸衍生物包括亚叶酸钙。
  9. 根据权利要求1-8中任一项所述的应用，其特征在于：
    所述药物还包含其他活性成分，所述其他活性成分包含维生素B、维生素C、烟酰胺、氧化锌、维生素A、维生素D、维生素E、维生素K、同型半胱氨酸、谷胱甘肽和牛磺酸中的至少一种；
    优选地，所述维生素B包含维生素B1、维生素B2、维生素B3、维生素B5、维生素B6、维生素B7和维生素B12中的至少一种；
    优选的，所述药物包括叶酸或其衍生物和烟酰胺；或所述药物包括叶酸或其衍生物和维生素E；或所述药物包括叶酸或其衍生物和维生素A以及维生素E。
  10. 根据权利要求1-9中任一项所述的应用，其特征在于：
    所述药物的剂型为儿童适用的剂型或成人适用的剂型；
    优选地，所述儿童包括出生28天内的新生儿、1岁以内的婴儿、1～6岁的幼儿、以及6～18岁的儿童；
    优选地，所述成人包括妊娠期的女性成年人、围产期的女性成年人和哺乳期的女性成年人。
  11. 根据权利要求1-9中任一项所述的应用，其特征在于：
    所述药物的剂型包括胶囊剂、片剂、微囊制剂、注射剂、栓剂、喷雾剂、散剂、软胶囊剂、滴丸、蜜丸、丸剂、颗粒剂、蜜炼膏剂、缓控释制剂、口服液体制剂、注射剂、咀嚼片、口腔片、透皮贴剂和泡腾片；
    或，
    所述药物的剂型包括胃肠道给药剂型或非胃肠道给药的剂型。
  12. 根据权利要求1-11中任一项所述的应用，其特征在于：所述叶酸或其衍生物在所述药物中的单位剂量为0.1mg～1g；
    优选地，所述叶酸或其衍生物在所述药物中的单位剂量为0.1mg、0.2mg、0.3mg、0.4mg、0.5mg、0.6mg、0.7mg、0.8mg、0.9mg或1.0mg；
    优选地，所述叶酸或其衍生物在所述药物中的单位剂量为0.2mg、0.4mg、0.8mg、或1.0mg。
  13. 胆道闭锁的诊断或辅助诊断标志物，其特征在于，
    所述诊断或辅助诊断标志物包含：
    a)叶酸；以及
    b)S100a8、S100a9、Nox2、IFN-γ中的至少一种；
    优选地，所述S100a8、S100a9、Nox2和IFN-γ包括蛋白质和/或mRNA。
  14. 根据权利要求13所述的诊断或辅助诊断标志物，其特征在于：
    所述诊断或辅助诊断标志物来自体液、血液、组织、细胞或排泄物；
    优选地，所述组织包括肝脏、肠道、胚胎肝脏、胚胎肠道；
    优选地，所述排泄物包括粪便、尿液；
    优选地，所述血液为全血、血清或血浆。
  15. 检测标志物的试剂在制备诊断或辅助诊断胆道闭锁的产品中的应用，其特征在于：
    所述标志物包含叶酸、S100a8、S100a9、Nox2、IFN-γ中的至少一种；
    优选地，所述S100a8、S100a9、Nox2、IFN-γ包括蛋白质和/或mRNA。
  16. 根据权利要求15所述的应用，其特征在于：
    所述产品包括试剂、试剂盒、基因芯片和蛋白质芯片；
    优选地，所述产品用于执行以下检测方法中的至少一种：
    (d1)比色法；(d2)化学发光法；(d3)原子吸收分光光度法；(d4)聚合酶链反应；(d5)微数字聚合酶链反应；(d6)荧光聚合酶链反应；(d7)环介导等温扩增反应；(d8)酶联免疫吸附测定法；(d9)核苷酸序列测序法；(d10)氨基酸序列测序法；(d11)变性梯度凝胶电泳；(d12)核酸分型芯片检测；(d13)高效液相色谱法；(d14)原位杂交；(d15)生物质谱法；(d16)高分辨率溶解曲线分析；(d17)单链构象异构多态分析；(d18)探针扩增阻滞突变系统分析。
  17. 根据权利要求15或16所述的应用，其特征在于：
    当标志物为S100a8、S100a9、Nox2和IFN-γ的mRNA时，所述产品含有检测所述标志物和/或内参mRNA的引物、和/或检测探针；
    优选地，当标志物为S100a8 mRNA，所述产品包括检测S100a8 mRNA的引物，所述引物的核苷酸序列如SEQ ID NO.1和SEQ ID NO.2所示；
    优选地，当标志物为S100a9 mRNA，所述产品包括检测S100a9 mRNA的引物，所述引物的核苷酸序列如SEQ ID NO.3和SEQ ID NO.4所示；
    优选地，当标志物为IFN-γmRNA，所述产品包括检测IFN-γmRNA的引物，所述引物的核苷酸序列如SEQ ID NO.5和SEQ ID NO.6所示；
    优选地，当标志物为Nox2 mRNA，所述产品包括检测Nox2 mRNA的引物，所述引物的核苷酸序列如SEQ ID NO.7和SEQ ID NO.8所示。
  18. 根据权利要求15-17中任一项所述的应用，其特征在于：
    当待测对象满足(c1)～(c5)中至少一项，则诊断为胆道闭锁疾病患者；
    (c1)叶酸相对于参考水平显著降低；
    (c2)S100a8相对于参考水平显著升高；
    (c3)S100a9相对于参考水平显著升高；
    (c4)IFN-γ相对于参考水平显著升高；
    (c5)Nox2相对于参考水平显著降低；
    所述参考水平为未患有胆道闭锁疾病的同年龄段受试者的水平；
    优选地，所述同年龄段受试者为健康人或胆总管囊肿患者。
  19. 根据权利要求15-18中任一项所述的应用，其特征在于：
    所述标志物来自体液、血液、组织、细胞或排泄物；
    优选地，所述组织包括肝脏、肠道、胚胎肝脏、胚胎肠道；
    优选地，所述排泄物包括粪便、尿液；
    优选地，所述血液为全血、血清或血浆。
  20. 根据权利要求15-19中任一项所述的应用，其特征在于：
    所述待测对象为成人或儿童；
    优选地，所述儿童包括出生28天内的新生儿、1岁以内的婴儿、1～6岁的幼儿、以及6～8岁的儿童；
    优选地，所述成人选自妊娠期的女性成年人、围产期的女性成年人或哺乳期的女性成年人。
  21. 一种试剂盒，包含检测标志物的试剂；
    所述标志物包含叶酸、S100a8、S100a9、Nox2、IFN-γ中的至少一种；
    优选地，所述标志物包含：
    a)叶酸；以及
    b)S100a8、S100a9、Nox2、IFN-γ中的至少一种。
PCT/CN2022/130161 2021-11-05 2022-11-05 叶酸在预防、诊断和治疗遗传性、感染性或过敏性疾病中的应用 WO2023078435A1 (zh)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH07291864A (ja) * 1994-04-27 1995-11-07 Yoshiaki Kajiyama 閉塞性黄疸治療剤
JPH11246408A (ja) * 1998-03-03 1999-09-14 Nippon Waisuredarii Kk 肝障害治療剤
CN1638751A (zh) * 2002-02-26 2005-07-13 默克阿泼洛发股份公司 叶酸盐在制备用于预防和治疗炎症和炎症相关性疾病,特别用于影响炎症标志物crp和saa的药物制剂中的用途
US20170290814A1 (en) * 2016-04-11 2017-10-12 Genfit Methods of treatment of cholestasis and fibrosis
CN109069648A (zh) * 2016-04-11 2018-12-21 基恩菲特公司 胆汁淤积性和纤维化疾病的治疗方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH07291864A (ja) * 1994-04-27 1995-11-07 Yoshiaki Kajiyama 閉塞性黄疸治療剤
JPH11246408A (ja) * 1998-03-03 1999-09-14 Nippon Waisuredarii Kk 肝障害治療剤
CN1638751A (zh) * 2002-02-26 2005-07-13 默克阿泼洛发股份公司 叶酸盐在制备用于预防和治疗炎症和炎症相关性疾病,特别用于影响炎症标志物crp和saa的药物制剂中的用途
US20170290814A1 (en) * 2016-04-11 2017-10-12 Genfit Methods of treatment of cholestasis and fibrosis
CN109069648A (zh) * 2016-04-11 2018-12-21 基恩菲特公司 胆汁淤积性和纤维化疾病的治疗方法

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