CN102041295A - Application of beta-1,4 galactosy transferase I in preparing liver cancer diagnostic preparation - Google Patents
Application of beta-1,4 galactosy transferase I in preparing liver cancer diagnostic preparation Download PDFInfo
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Abstract
The invention belongs to the fields of molecular biology and medicine, and relates to an application of beta-1,4 galactosy transferase I in preparing a liver cancer diagnostic preparation. In the invention, the activity of the beta-1,4 galactosy transferas I is utilized as a liver cancer diagnostic index. The activity enhancement of the beta-1,4 galactosy transferase I refers to the increase of the expression quantity of the beta-1,4 galactosy transferase I, or the increase of the quantity of beta-1,4 galactoside bonds in a sample. The activity index of the beta-1,4 galactosy transferase I is used for liver cancer primary screening, diagnosis and prognoses judgement, and prognoses judgement and relapse diagnose after liver cancer operation, and is peculiar and sensitive, and is high efficient and simple; by utilizing the detection method provided by the invention, in vitro reaction is carried out on only a quite small amount of extracted hepatic tissue or blood samples of a patient, thus the pain of the patient and the burden for the family of the patient are greatly relieved. In addition, the index also can complement the advantages with other liver cancer tags such as tetrapeptide protein and the like to serve as a reference of liver cancer diagnosis.
Description
Technical field
The invention belongs to molecular biology and medical field, be specifically related to the application of β-1,4 galactosyltransferase I in preparation diagnosing cancer of liver preparation.More specifically, relate to β-1,4 galactosyltransferase I as liver cancer primary dcreening operation, diagnosis and prognosis judgement and operation of liver cancer after an index of prognosis judgement and relapse diagnosis aspect.
Background technology
Primary hepatocarcinoma (abbreviation liver cancer) is one of China's common malignancy, and its tumor incidence is the 3rd the male sex, is only second to the cancer of the stomach and the esophageal carcinoma, and the women then is the 4th.The annual newly-increased patient 110,000 of China, crowd's sickness rate is 23.7/10 ten thousand people.Because liver cancer onset concealment, in having belonged to mostly when the patient goes to a doctor, late period, to add and merge liver cirrhosis rate height and the high multiple factor of recurrence after operation rate, result of treatment is relatively poor, the mortality ratio height.Therefore seem particularly important for prognosis judgement and relapse diagnosis behind primary dcreening operation, diagnosis and the prognosis judgement and the operation of liver cancer of liver cancer, present detection index has certain limitation, presses for to find new diagnosis index.Studies confirm that in a large number people and animal model liver cancer cell are compared protein with normal liver cell and the lipid glycosylation there are differences.The glycoprotein that some glycosylation changes has been applied to the diagnosis of human liver cancer, by stages and prognosis evaluation; Some polysaccharid derivatives have been applied to the assisting therapy of liver cancer simultaneously.
Unusual glycosylation in the liver cancer cell mainly comprises the change of glycoprotein and glycolipid component.Wherein glycoprotein be protein and oligonucleotide chain by covalently bound mixture, mainly contain two kinds of mode of connection: a kind of hydroxyl oxygen for Serine in sugar chain and the peptide chain or Threonine links to each other, and is called the O-sugar chain; Another kind is that sugar chain links to each other with the amido linkage of l-asparagine in the peptide chain, is called the N-sugar chain.Wherein more with the mode of connection of N-sugar chain, also closer with the relation of tumour.Van Beek finds during glycoprotein on rat hepatoma cell, tire liver and the newborn rat liver plasma membrane of more different grade malignancies, all liver cancer cells all have the glycoprotein that contains the volume Fucose to occur, and it is irrelevant with grade malignancy, tire liver and newborn rat liver cell then do not have this kind change, it is the phenomenon of a malignant tumour that the glycosylation of proof glycoprotein changes, and irrelevant with the normal growth of cell.The phenomenon that the glycoprotein candy chain molecule of this tumour cell increases is so-called Warren-Glick phenomenon.Studies show that further the relative molecular mass increase of sugar chain is because the sugar chain ramose increases, and wherein sees with sialic increasing on the sugar chain especially more.N-glycan change type common in the liver cancer can reduce 9 kinds of listed situations of table 1.
The glycosylation of glycoprotein needs the participation of glycosyltransferase, so the change of sugar chain structure is inevitable relevant with the activity change of transferring enzyme in the liver cancer cell glycoprotein.The consistence that enzyme activity changes and the N-glycan structures changes is existing to be experimental results demonstrate, as when nitrosamine is brought out rat liver cancer, divide type N-acetyl-glucosamine (GlcNAc) and antenna number in the N-glycan of liver neutral and alkali Phosphoric acid esterase (ALP) equally and increase to increase with the vigor of GlcNAc transferring enzyme (GlcNAcT)-III and V respectively and be consistent.The N-glycan of alpha-fetoprotein (AFP) has more C2C2 in the strain of HepG2 liver cancer, 6 triantennaries and core Fuc, and its GlcNAcT-V and α 1, (α 1, and vigor 6-FucT) is all higher for the 6-fucosyl transferase.The AFP of HuH-7 liver cancer strain only contains few type GlcNAc and C2C2 of dividing equally, 6 triantennary structures, and its GlcNAcT-III and V are all lower.The E-cadherin (E-cadherin) on high metastatic cancer cell surface of discovering transfection GlcNAc T-III is from the cell surface minimizing that comes off, the invasion and attack of cell and transfer ability reduce, structural analysis shows that the type GlcNAc that divides equally on the E-cadherin glycan increases, and β 1,6 connect the then minimizing of GlcNAc, and the vigor of prompting GlcNAcT-V is suppressed.Therefore, GlcNAcT-V can regard as respectively and short metastases or the relevant glycosyltransferase of anti metastasis with GlcNAcT-III.In addition, our β 1,4 galactosyltransferase I (GalT I) that studies show that can make the β 1,6 on the human hepatoma cell strain N-glycan connect the GlcNAc increasing expression, thereby promotes the growth of tumour cell and invasion and attack and form in the intravital focus of nude mice.
The type that glycoprotein N-glycan changes in table 1. liver cancer
And glycolipid is lipid and oligonucleotide chain covalent attachment and the mixture that forms can be divided into sphingoglycolipid and glyceroglycolipid two big classes.Studies show that in recent years, liver cell are cancerated and are often followed in the process sphingoglycolipid particularly with the change of sialic Sphingolipids,sialo.In analysis to normal liver tissue and liver cancer tissue Sphingolipids,sialo component, find that the abundant neural straw glycosides of single sialic acid fat 3 (GM3) of content obviously reduce in the healthy tissues in liver cancer cell, and the less neural straw glycosides of two sialic acids fat 3 (GD3) of content about 10 times have been increased in the healthy tissues in liver cancer tissue.
SLeX is a kind of pentasaccharides structural unit that contains sialic acid and Fucose, and in existence and glycoprotein and the glycolipid, it is the main part of ELAM-1.21% liver cancer expression sLeX is pointed out in research, and non-cancer liver cell is not expressed sLeX, and liver cancer is expressed positive person more than the liver cancer patient of not accompanying transfer with transferrer sLeX in the liver, and liver cancer mortality also is proportionate with the sLeX expression.Apparently higher than non-cancer hepatic tissue, and the expression of IV, VI FucT is relevant with the metastatic phenotype of liver cancer in cancerous tissue for one of the main synthetase series of sLeX α 1,3-FucT.
Glycoprotein on the cytolemma, glycolipid all have the important physical function, in case sugar chain changes, will cause dysfunction to cause serious consequence.Intercellular adhesion and identification depend on the identification to sugar chain, and the contact inhibition of regulating the cell normal growth just depends on intercellular identification.When sugar chain structure changed, the crosslinked obstacle of iuntercellular took place in the time of can making cell growth contact in the particularly shortening of glycolipid sugar chain, and iuntercellular can not transmit information, thereby causes the disappearance of contact inhibition.Sphingolipids,sialo also have the function of regulating growth factor receptors, thus the growth of control cell.As GM3 the function that is adjusted to bfgf receptor is arranged, can be to make fibrocellular propagation.When malignant change of cell, because the change of sugar chain structure causes identification and cell cycle regulation dysfunction between the cell, make cell be property out of control growth, have a kind of trend of infinite multiplication.The sugar chain of surface of cell membrane is also being regulated intercellular adhesive attraction.Its essence of adhesion molecule on the cytolemma mostly is glycoprotein, when glycoprotein generation glycosylation, when particularly sialylated, intercellular adhesion is changed, thereby help the transfer of liver cancer cell.
Glycoprotein, glycolipid also are the components of many membrane antigen determinants.When canceration of hepatic cell,, make liver cancer cell escape the attack of host immune system because the change of sugar chain structure causes the change of membrane antigen.And because the tumour cell metabolism changes the synthetic of the novel glycoprotein cause, glycolipid, be exactly the integral part that constitutes tumour specific antigen probably.
The detection of liver cancer marker and analysis help that diagnosing cancer of liver, observation of curative effect, postoperative are followed up a case by regular visits to, recurrence or transfer monitoring.Clinical liver cancer marker commonly used mostly is saccharide complex at present, is many with glycoprotein especially.
The clinical liver cancer marker commonly used of table 2
Annotate: the most frequently used several liver cancer markers that are that underscore " _ _ _ _ " is arranged in the table.
AFP belongs to embryo's property albumen, but diagnosing liver cancer is had limitation.Because of AFP in the optimum hepatopathy of part and some malignant tumours also raises, and part liver cancer patient AFP continues the lower concentration positive all the time.The total AFP of serum contains: AFP-L1 accounts for its major portion from optimum hepatopathy; AFP-L2 is from the pregnant woman; AFP-L3 then is a liver-cancer cell specific.Total AFP is because of different with the phytohaemagglutinin affinity on the sugar chain The Nomenclature Composition and Structure of Complexes, adopt electrophoretic method to isolate LcA (LCA) in conjunction with AFP-L3, its normal value is 10%～15%, if＞15%, even total AFP only slightly raises, or askiatic evidence, all pointed out and developed into the HCC possibility, should tightly monitor and regular follow-up.The susceptibility of AFP-L3 diagnosing liver cancer is 96.9%, and specificity is 92.0%, and accuracy is 95.5%, and small liver cancer is had higher specificity, and is meaningful in prognosis in hcc.
AFU is a kind of new diagnosis marker of primary hepatocarcinoma, is distributed widely in each histocyte lysosome of human body and the body fluid, mainly participates in the katabolism of the macromolecular substance such as various glycolipids, glycoprotein, mucopolysaccharide of kukersite algae glycosyl.The AFU activity not only is significantly higher than normal control in the Patients with Primary serum, and is significantly higher than secondary liver cancer, bile duct cell, malignant mesothe, malignant hemangioendothelioma, liver cirrhosis, congenital cyst of liver and other optimum hepatic space occupying lesion.Therefore, diagnosis has higher susceptibility and specificity to serum AFU to primary hepatocarcinoma, and especially AFP diagnostic value negative and minicell liver cancer is bigger.
The change that utilizes lectin to analyze N-glycan in some liver cancer excretory blood plasma or other body fluid protein has been used as one of diagnostic means of liver cancer.Derive from very disease of primary hepatocarcinoma, other malignant tumours or liver property as the above-mentioned alpha-fetoprotein (AFP) that increases among the patients serum of how much can distinguishing with LcA (LCA) analysis core Fucose.And for example deriving from the transferrin (Tf) of liver and the N-glycan of liver type alkaline phosphatase isoenzyme (L-ALP) in the normal serum all is two antenna structures, and the antenna number of N-glycan increases among the Tf of schistosomiasis japonica blood serum and the ALP, the avidity of coagulating plain (DSA) with thorn apple increases, and available enzyme mark DSA or solid phase DSA affinity post are identified.These means have been used to the supplemental diagnostics of AFP negative primary liver cancer.
Saccharide complex not only is widely used in the laboratory examination of liver cancer, also is used to the other technologies of diagnosing cancer of liver in recent years gradually.The part that utilizes radioisotope labeling combines with corresponding specific receptors and carries out organ or tissue's video picture, is the nuclear medicine image new technology that development in recent years is got up.99mTc-semi-lactosi-Xin sugar albumin (99mTc-NGA), as the designate similar thing of HBP (HBP) native ligand, it can be optionally combines with HBP on the liver plasma membrane, and the evaluation of hepatocyte function is had unique clinical value.
Although in the clinical practice at present multiple liver cancer marker has been arranged, seek the New Set that specificity is good, highly sensitive, detection is convenient or with low cost, be still the heat subject of this area.
Summary of the invention
The new purposes that the purpose of this invention is to provide β-1,4 galactosyltransferase I is specifically related to the application of β-1,4 galactosyltransferase I in preparation diagnosing cancer of liver preparation.
The invention provides β-1,4 galactosyltransferase I (beta-1,4galactosyltransferase, β 1,4GalT I) application in diagnosing cancer of liver, particularly, be as the diagnosing cancer of liver index with β-1,4 galactosyltransferase I activity in hepatic tissue or the blood sample; Wherein, β-1,4 galactosyltransferase I increased activity is meant that β-1, the 4 galactosyltransferase I expression amount of tissue samples increases the perhaps increase of the quantity of β in the sample-1,4 semi-lactosi glycosidic bond.
According to statistics of the present invention, β in the liver cancer sample-1,4 galactosyltransferase I activity increases by 50% compared with the control at least, and can reach preferably increases by 70%, 100%, so that increase more than 2 times.
Therefore, can be with β-1,4 galactosyltransferase I increased activity in hepatic tissue and the blood as the diagnosis index of liver cancer primary dcreening operation, also can be with β-1,4 galactosyltransferase I increased activity in hepatic tissue and the blood as the diagnosis index of recurrence of PHC.
Sxemiquantitative RT-PCR shows β 1, and 4GT I expression amount in liver cancer tissue is more than 2 times of expression amount in the normal liver tissue, and liver cancer tissue epicyte protein galactosylation generally raises.And the galactosylation of hepatocarcinoma patient postoperative serum protein almost disappears, and the galactosylation of the hepatocarcinoma patient serum of postoperative recurrence obviously increases, even reaches the preceding level of art.
β-1,4 galactosyltransferase I (beta-1,4 galactosyltransferase; EC 2.4.1.38) (being called for short β 1,4GT I) is a kind of glycosyltransferase of being cloned the earliest.It is positioned golgi body, can form by catalysis Gal β 1 → 4GlcNAC glycosidic link, and synthesizing of involved in sugar mixture participates in the formation of the many antennas of N-Glycan.The glycosylation substrate of existing confirmed β-1,4 galactosyltransferase I comprises EGFR, immunoglobulin (Ig) etc.Being expressed in the kinds of tumors of β-1,4 galactosyltransferase I increases, and relevant with the grade malignancy of tumour.The expression that changes β-1,4 galactosyltransferase I influences the invasive ability of melanoma and lung carcinoma cell.
In the sequence table of the present invention, SEQ ID NO.1 and SEQ ID NO 2 are respectively base sequence and the aminoacid sequence of β-1,4 galactosyltransferase I.Its nucleotide coding sequence of the β of indication of the present invention-1,4 galactosyltransferase I comprises SEQ ID NO 1 and degenerate sequence thereof.The degenerate sequence of SEQ ID NO 1 is meant, in the SEQ ID NO.1 sequence, having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, so be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.2 of also encoding out with SEQ ID NO.1 nucleotide sequence homology.The β of indication of the present invention-1,4 galactosyltransferase I comprises that also SEQ ID NO 2 similarities are more than 75% and albumen or polypeptide with β-1,4 galactosyltransferase I function.
Among the present invention, β-1,4 galactosyltransferase I increased activity comprises that expression amount increases the increase with the zymoprotein catalytic activity of unit volume or quality.
β in human liver tissue and the blood-1,4 galactosyltransferase I (β 1 for beta-1,4galactosyltransferase, 4GalT I) increased activity can be used as the index that liver cancer primary dcreening operation, diagnosis and prognosis are judged.
β in human liver tissue and the blood-1,4 galactosyltransferase I (β 1 for beta-1,4galactosyltransferase, 4GalT I) increased activity can be used as the index of prognosis judgement and relapse diagnosis behind the operation of liver cancer.
RCA-1 lectin staning analyzes demonstration and compares with healthy tissues, and liver cancer tissue epicyte protein galactosylation has the trend that generally raises.And RCA-1 lectin blot analysis shows that the galactosylation of hepatocarcinoma patient postoperative serum protein obviously reduces, and the galactosylation of the hepatocarcinoma patient serum of postoperative recurrence obviously increases.Liver cancer tissue RCA-I stained positive rate has been compared statistical significance (P＜0.05) with normal cancer beside organism.The galactosylation of hepatocarcinoma patient postoperative serum protein obviously reduces, and the galactosylation of the hepatocarcinoma patient serum of postoperative recurrence obviously increases, compare with the galactosylation of hepatocarcinoma patient postoperative serum protein, the serum protein galactosylation of hepatocarcinoma patient and postoperative recurrence thereof also has statistical significance (P＜0.05).Blank adds 0.12mol/L when replacing lectin or adding lectin with sheep blood serum semi-lactosi competition suppresses no positive signal.
The present invention also provides above-mentioned β-1,4 galactosyltransferase I detection methods, the RT-PCR and the qRT-PCR that comprise (1) hepatic pathology tissue, (2) hepatic pathology organize RCA-1 lectin staining and the proteic RCA-1 lectin of (3) patients serum blot.
The β-1 of detection hepatic tissue provided by the invention or blood sample, the active method of 4 galactosyltransferase I comprises β-1, the 4 galactosyltransferase I expression amount that detects the hepatic tissue sample, the perhaps quantity of β-1,4 galactoside in hepatic tissue or the blood sample.
Among the present invention, can utilize RT-PCR or qRT-PCR method to detect β-1, the 4 galactosyltransferase I expression amount of hepatic tissue sample.
RT-PCR is the abbreviation of reverse transcription RCR (reverse transcription PCR).
Reverse transcription PCR claims that perhaps (reverse transcript ion-PCR RT-PCR), is the distortion of a kind of widespread use of polymerase chain reaction (PCR) to reverse transcription PCR.In RT-PCR, a RNA chain is reversed record becomes complementary DNA, carries out DNA cloning as template by PCR again.
Be transcribed into complementary DNA (cDNA) by a RNA single strand and be called " reverse transcription ", finish by the archaeal dna polymerase (reversed transcriptive enzyme) of dependenc RNA.Subsequently, another of DNA chain is finished by the archaeal dna polymerase of deoxynucleotide primer and dependence DNA, with each circulation multiplication, promptly common PCR.Original RNA template is stayed complementary DNA by RNA enzyme H degraded.
The instant real quantitatively PPCR of qRT-PCR belongs to a kind of of quantitative PCR (Q-PCR), is the quantitative analysis that DNA is carried out on the basis with the amount of amplification of DNA in the certain hour.
The quantitative use fluorescent pigment of real time PCR has two kinds of methods at present.A kind of is to insert special fluorescent pigment in ds DNA; A kind of a kind of fluorescent probe (probe) that can combine of another kind of use with specific few nucleotide sequence in the amplification dna sequence dna.
Thereby so-called real-time fluorescence quantitative PCR is exactly to realize starting template quantitatively and is qualitatively analyzed by the real-time detection to each circulation products fluorescent signal in the pcr amplification reaction.In the real-time fluorescence quantitative PCR reaction, introduced a kind of fluorescence chemical material, along with the carrying out of PCR reaction, the PCR reaction product constantly adds up, and fluorescence signal intensity also equal proportion increases.Every through a circulation, collect a fluorescence intensity signals, we just can pass through the variation of fluorescence intensity variation monitoring product amount like this, thereby obtain an amplified fluorescence curve.
QRT-PCR is made up of three steps:
1. reverse transcription: rely on ThermoScript II the RNA reverse transcription is become cDNDA;
2. amplification: with the method amplification cDNA of PCR;
3. detect: the product of real-time detection and quantitative amplification.
PCR generally includes the step of following two aspects:
(1) pre-sex change:
Destroy the more difficult destructive secondary structure that may exist among the DNA.Make the abundant sex change of DNA, reduce of the influence of DNA complex construction, be beneficial to the better and template combination of primer amplification, particularly for the dna profiling in genome source, had better not stingy this step.In addition, use in the reaction of warm start Taq enzyme, also can activate the Taq enzyme, thereby make the PCR reaction smooth at some.
(2) circulation is extended in sex change--annealing--:
1. the sex change of template DNA: template DNA dissociates template DNA double-stranded DNA double-stranded or that form through pcr amplification after being heated to 93 ℃ of left and right sides certain hours, makes it to become strand, so that it combines with primer, for the lower whorl reaction is prepared;
2. the annealing (renaturation) of template DNA and primer: template DNA is after heat denatured becomes strand, and temperature is reduced to about 55 ℃, and primer combines with the complementary sequence pairing of template DNA strand;
3. the extension of primer: dna profiling--primer binding substances is a reaction raw materials with dNTP under the effect of TaqDNA polysaccharase, and target sequence is a template, by base pairing and semiconservative replication principle, synthesizes new and a template DNA chain complementary semiconservative replication chain.
Preferably, among the present invention, the β of hepatic tissue sample-1, the detection method of 4 galactosyltransferase I expression amounts can be carried out according to following method: the RNA that extracts the hepatic tissue sample, reverse transcription becomes cDNA then, the nucleotide coding sequence of amplification β-1,4 galactosyltransferase I, products therefrom increases more than the β that is the hepatic tissue sample-1, the 4 galactosyltransferase I expression amount of contrast.
Among the present invention, the detection method of β in the hepatic tissue sample-1,4 galactoside bond number amount is: the hepatic tissue sample is made paraffin section, the dewaxing entry; Add H
2O
2Perhaps methyl alcohol is blocked endogenic peroxidase, adds avidin and blocks endogenic vitamin H; Sialidase digestion, sealing adds vitamin H-ricinus agglutinin-I overnight incubation then; Avidin-vitamin H that the adding mark is crossed is hatched, colour developing; Control group replaces vitamin H-ricinus agglutinin-I with sheep blood serum, perhaps adds the semi-lactosi competition and suppress when adding vitamin H-ricinus agglutinin-I.
In the detection method of β in the above-mentioned blood sample-1,4 galactoside bond number amount, can adopt horseradish peroxidase to carry out mark, the diaminobenzidine colour developing.
Among the present invention, β in the blood sample-1, the detection method of 4 galactoside bond number amounts is: extract the serum protein in the blood sample, western blot transfers to pvdf membrane, and sealing is spent the night, and hatches with ricinus agglutinin-I that mark is crossed, wash film, detect then ricinus agglutinin-I in conjunction with quantity, the increase in conjunction with quantity of ricinus agglutinin-I is the increase of β in the hepatic tissue sample-1,4 galactoside bond number amount.
In the detection method of β in the above-mentioned blood sample-1,4 galactoside bond number amount, can adopt horseradish peroxidase to carry out mark, the ECL chemiluminescence system detect ricinus agglutinin-I in conjunction with quantity.
(ricinus communis agglutinin-I RCA-I) is a class carbohydrate-binding protein to ricinus agglutinin-I, can discern N-connecting-type sugar-chain end β-1,4-semi-lactosi glycosidic bond (Gal β 1 → 4GlcNAc) structure single-mindedly.The avidin-avidin claims again " avidin ".DAB is a diaminobenzidine.Electrochemiluminescence (ECL) system, and electrochemiluminescence (electrochemiluminescence, ECL).
The case sample comes from shanghai Medicine institute of Fudan University.
The immunohistochemical concrete steps of lectin are: according to document [Shen A, Zhu D, Ding F, et al.Increased gene expression of beta-1,4-galactosyltransferase I in rat injured sciatic nerve[J] .J Mol Neurosci, 2003,21 (2): 103-110.] report and carry out the lectin immunohistochemical staining: get glioma paraffin sections at different levels, the dewaxing entry, 3%H
2O
2/ methyl alcohol, 37 ℃ of 10min are to block endogenic peroxidase.Add avidin (25g/L) room temperature 30min, to block endogenic vitamin H.37 ℃ of 5h of sialidase (0.03mg/L) digestion are to remove outside sialic acid chain.1%BSA behind 37 ℃ of 2h, adds Biotin-RCA-I (10mg/L) 4 ℃ and spends the night.Add avidin-biotin-HRP (horseradish peroxidase) mixture (1: 150).Conventional DAB colour developing.Blank adds 0.2mol/L when replacing lectin or adding lectin with sheep blood serum semi-lactosi competition suppresses, and all the other steps are identical.After colour developing finished, Hematorylin was redyed.Experiment repeats 3 times under the same conditions.
RCA-I lectin blot detects the concrete steps of organizing galactosylation to change: pvdf membrane is positioned in the 25ml confining liquid (150mmol/LNaCl, 10mmol/L TrisHCl pH8.0,0.1%Tween-20,5% skimmed milk), and 4 ℃ of sealings are spent the night.The RCA-I of 10g/L horseradish peroxidase-labeled is hatched 1h for 37 ℃.(150mmol/LNaCl, 10mmol/L TrisHCl pH8.0 0.1%Tween-20), wash 15min 3 times to TTBS.The ECL chemiluminescence system detects, compressing tablet, punching.
Pvdf membrane (claiming Positively charged nylon again) (Polyvinylidene fluoride, PVDF membrane).The pvdf membrane of tool chemoresistance is used for albumen research, because pvdf membrane has very high binding ability, and repeatedly still can avoid tearing and embrittlement behind transfer and the mark, and it is always as western blot hybridization film for a long time.Though the efficient of PDVF embrane-associated protein less than the nitrocellulose filter height, because the stable, corrosion-resistant of it makes it become protein sequencing ideal articles for use, is used till today always.Pvdf membrane is the same with nitrocellulose filter, can carry out various dyeing and chemiluminescence detection, and the very wide scope of application is also arranged.This pvdf membrane, sensitivity, resolving power and the protein affinity film than conventional under hand work is all high, is very suitable for the detection of low molecular weight protein (LMWP).But pvdf membrane must soak saturated 1-5 second with pure methyl alcohol before using.
The present invention relates in human liver's disease patient, carry out an index of prognosis judgement and relapse diagnosis behind liver cancer primary dcreening operation, diagnosis and prognosis judgement and the operation of liver cancer, this diagnosis index is β in human body hepatic tissue and the blood-1,4 galactosyltransferase I (beta-1,4galactosyltransferase, β 1,4GalT I) increased activity.Its detection method comprises the RT-PCR and the qRT-PCR of hepatic pathology tissue, and hepatic pathology is organized RCA-1lectin staining and the proteic RCA-1lectin blot of patients serum.
With β of the present invention-1,4 galactosyltransferase I activity index are used for prognosis judgement and relapse diagnosis behind liver cancer primary dcreening operation, diagnosis and prognosis judgement and the operation of liver cancer, special sensitivity, high-efficient simple, and detection method of the present invention only need be extracted the very a spot of hepatic tissue of patient or blood sample carries out vitro reactions, has alleviated patient's misery and family members' burden thereof greatly.In addition, β of the present invention-1,4 galactosyltransferase I increased activity index can also be had complementary advantages with other diagnosing cancer of liver mark such as first peptide protein, the foundation of making a definite diagnosis as liver cancer.
Description of drawings
Fig. 1: sxemiquantitative RT-PCR shows β 1, and 4GTI trace in normal liver tissue is expressed high expression level in liver cancer tissue.
Fig. 2: RCA-1lectin staning analyzes demonstration and compares with healthy tissues, and liver cancer tissue epicyte protein galactosylation has the trend that generally raises.
Fig. 3: RCA-1lectin blot analyzes the galactosylation that shows hepatocarcinoma patient postoperative serum protein and obviously reduces, and the galactosylation of the hepatocarcinoma patient serum of postoperative recurrence obviously increases."+" number preceding or postoperative recurrence of expression art, "-" number expression postoperative.
Embodiment
The sxemiquantitative RT-PCR of embodiment 1 hepatic pathology tissue
1mg hepatic pathology tissue adds the homogenate of 1ml Trizol reagent, and room temperature is placed 5-10min, and collection organization's lysate is managed in Eppendorf, adds chloroform 0.2ml, jolting mixing 15 seconds, and room temperature is placed 10min.4 ℃, the centrifugal 10min of 12000g gets the new Eppendorf pipe of supernatant to, adds the Virahol of 500 μ l, and mixing is after room temperature is placed 10min.4 ℃, less than the centrifugal 10min of 12000g, abandon supernatant, precipitate washing with alcohol with 75%, room temperature leaves standstill 10min.After centrifugal, abandon ethanol to the greatest extent, drying at room temperature is dissolved with 20 μ l0.5%SDS.OD 260/280 colorimetric estimation RNA concentration.
1) by following composition preparation inverse transcription reaction liquid.
2) carry out reverse transcription reaction by following condition.
3) by following composition preparation PCR reaction solution.
4) above-mentioned reaction solution is joined in the PCR reaction tubes after reverse transcription finishes, gently mixing.Carry out the PCR reaction by following condition.
5) after reaction finishes, extract reaction solution and carry out agarose gel electrophoresis.
The result is as shown in Figure 1: sxemiquantitative RT-PCR shows β 1, and 4GT I trace in normal liver tissue is expressed high expression level in liver cancer tissue.
Embodiment 2 hepatic pathology tissue slice RCA-I dyeing
1) 56 ℃ of roasting sheets of paraffin section spend the night, and immerse dewaxing in 30 minutes in 37 ℃ of dimethylbenzene while hot, change fresh dimethylbenzene 10 minutes again over to, the entry of gradient alcohol.
2) PBS embathes 2 times; Add 3%H
2O
2/ methyl alcohol, 37 ℃, 10 minutes, to block endogenic peroxidase.
3) PBS embathes 2 times; Add avidin (25mg/ml), room temperature, 30 minutes, to block endogenic biotin.
4) PBS embathes 3 times; Sialidase (0.03 μ g/ml) digestion, 37 ℃, 5 hours to remove outside sialic acid chain.
5) after PBS embathes 3 times, add 37 ℃ of confining liquids, 2 hours (confining liquid: 1%BSA, 0.1%Triton-X100, pH7.4,0.01M PBS preparation).
6) add Biotin-RCA-I (Vector, dilution in 1: 2000,10 μ g/ml), 4 ℃, spend the night.
7) PBS embathes 3 times; Add Avidin-Biotin-HRP complex (ABC, Vector face with preparing half an hour).0.03%H
2The O/0.04%DAB colour developing, conventional dehydration, transparent, mounting, observations under the light microscopic.
8) control experiment of immunohistochemical methods replaces one anti-ly and add a semi-lactosi competition that adds 0.2M when anti-simultaneously and suppress with sheep blood serum, and all the other steps are identical.Test triplicate under the same conditions.
The RCA-I lectin of vitamin H (biotin) mark, the ABC test kit is purchased in Vector; The anti-mouse IgG of biotin mark purchases the Cruz in Santa.
The result is as shown in Figure 2: RCA-1lectin staning analyzes and shows and compares with healthy tissues, and liver cancer tissue epicyte protein galactosylation has the trend that generally raises.Statistics shows that liver cancer tissue epicyte protein galactosylation can reach about 2 times of healthy tissues.
The proteic RCA-1 lectin of embodiment 3 patients serums blot
1) gets the patients serum and be put in 4 ℃ of preservations.Get 50 μ l and add 2 * SDS Loading Buffer, boiled 10 minutes.
2)Western?Blot。
3) pvdf membrane is put as for 85 ℃ in 25mM sulfuric acid, handled after 1 hour, TBST gives a baby a bath on the third day after its birth inferior.
4) the 25ml confining liquid (150mM NaCl, 10mM Tris HCl PH8.0,0.1%Tween-20,1%BSA) in, 4 ℃ of sealings are spent the night.
5) RCA-1 of 10 μ g/ml horseradish peroxidase-labeled was hatched 1 hour for 37 ℃.(0.1%Tween-20), 15min washes 3 times TTBS for 150mMNaCl, 10mMTris HCl PH8.0.The ECL chemiluminescence system detects.Compressing tablet, punching.
The result is as shown in Figure 3: RCA-1lectin blot analyzes the galactosylation that shows hepatocarcinoma patient postoperative serum protein and obviously reduces, and be close to disappearance, and the galactosylation of the hepatocarcinoma patient serum of postoperative recurrence increases obviously.
All documents of mentioning in the present invention are just quoted as a reference separately as each piece document all in this application as a reference.Should be understood that in addition read this explanation above-mentioned tell about content after, those skilled in the art can make various changes or modifications the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
<210>1
<211>1197
<212>DNA
＜213〉mankind
<220>
<221>CDS
<222>(1)..(1197)
<223>
<400>1
atg?agg?ctt?cgg?gag?ccg?ctc?ctg?agc?ggc?agc?gcc?gcg?atg?cca?ggc 48
Met?Arg?Leu?Arg?Glu?Pro?Leu?Leu?Ser?Gly?Ser?Ala?Ala?Met?Pro?Gly
1 5 10 15
gcg?tcc?cta?cag?cgg?gcc?tgc?cgc?ctg?ctc?gtg?gcc?gtc?tgc?gct?ctg 96
Ala?Ser?Leu?Gln?Arg?Ala?Cys?Arg?Leu?Leu?Val?Ala?Val?Cys?Ala?Leu
20 25 30
cac?ctt?ggc?gte?acc?ctc?gtt?tac?tac?ctg?gct?ggc?cgc?gac?ctg?agc 144
His?Leu?Gly?Val?Thr?Leu?Val?Tyr?Tyr?Leu?Ala?Gly?Arg?Asp?Leu?Ser
35 40 45
cgc?ctg?ccc?caa?ctg?gtc?gga?gtc?tcc?aca?ccg?ctg?cag?ggc?ggc?tcg 192
Arg?Leu?Pro?Gln?Leu?Val?Gly?Val?Ser?Thr?Pro?Leu?Gln?Gly?Gly?Ser
50 55 60
aac?agt?gcc?gcc?gcc?atc?ggg?cag?tcc?tcc?ggg?gag?ctc?cgg?acc?gga 240
Asn?Ser?Ala?Ala?Ala?Ile?Gly?Gln?Ser?Ser?Gly?Glu?Leu?Arg?Thr?Gly
65 70 75 80
ggg?gcc?cgg?ccg?ccg?cct?cct?cta?ggc?gcc?tcc?tcc?cag?ccg?cgc?ccg 288
Gly?Ala?Arg?Pro?Pro?Pro?Pro?Leu?Gly?Ala?Ser?Ser?Gln?Pro?Arg?Pro
85 90 95
ggt?ggc?gac?tcc?agc?cca?gtc?gtg?gat?tct?ggc?cct?ggc?ccc?gct?agc 336
Gly?Gly?Asp?Ser?Ser?Pro?Val?Val?Asp?Ser?Gly?Pro?Gly?Pro?Ala?Ser
100 105 110
aac?ttg?acc?tcg?gtc?cca?gtg?ccc?cac?acc?acc?gca?ctg?tcg?ctg?ccc 384
Asn?Leu?Thr?Ser?Val?Pro?Val?Pro?His?Thr?Thr?Ala?Leu?Ser?Leu?Pro
115 120 125
gcc?tgc?cct?gag?gag?tcc?ccg?ctg?ctt?gtg?ggc?ccc?atg?ctg?att?gag 432
Ala?Cys?Pro?Glu?Glu?Ser?Pro?Leu?Leu?Val?Gly?Pro?Met?Leu?Ile?Glu
130 135 140
ttt?aac?atg?cct?gtg?gac?ctg?gag?ctc?gtg?gca?aag?cag?aac?cca?aat 480
Phe?Asn?Met?Pro?Val?Asp?Leu?Glu?Leu?Val?Ala?Lys?Gln?Asn?Pro?Asn
145 150 155 160
gtg?aag?atg?ggc?ggc?cgc?tat?gcc?ccc?agg?gac?tgc?gtc?tct?cct?cac 528
Val?Lys?Met?Gly?Gly?Arg?Tyr?Ala?Pro?Arg?Asp?Cys?Val?Ser?Pro?His
165 170 175
aag?gtg?gcc?atc?atc?att?cca?ttc?cgc?aac?cgg?cag?gag?cac?ctc?aag 576
Lys?Val?Ala?Ile?Ile?Ile?Pro?Phe?Arg?Asn?Arg?Gln?Glu?His?Leu?Lys
180 185 190
tac?tgg?cta?tat?tat?ttg?cac?cca?gtc?ctg?cag?cgc?cag?cag?ctg?gac 624
Tyr?Trp?Leu?Tyr?Tyr?Leu?His?Pro?Val?Leu?Gln?Arg?Gln?Gln?Leu?Asp
195 200 205
tat?ggc?atc?tat?gtt?atc?aac?cag?gcg?gga?gac?act?ata?ttc?aat?cgt 672
Tyr?Gly?Ile?Tyr?Val?Ile?Asn?Gln?Ala?Gly?Asp?Thr?Ile?Phe?Asn?Arg
210 215 220
gct?aag?ctc?ctc?aat?gtt?ggc?ttt?caa?gaa?gcc?ttg?aag?gac?tat?gac 720
Ala?Lys?Leu?Leu?Asn?Val?Gly?Phe?Gln?Glu?Ala?Leu?Lys?Asp?Tyr?Asp
225 230 235 240
tac?acc?tgc?ttt?gtg?ttt?agt?gac?gtg?gac?ctc?att?cca?atg?aat?gac 768
Tyr?Thr?Cys?Phe?Val?Phe?Ser?Asp?Val?Asp?Leu?Ile?Pro?Met?Asn?Asp
245 250 255
cat?aat?gcg?tac?agg?tgt?ttt?tca?cag?cca?cgg?cac?att?tcc?gtt?gca 816
His?Asn?Ala?Tyr?Arg?Cys?Phe?Ser?Gln?Pro?Arg?His?Ile?Ser?Val?Ala
260 265 270
atg?gat?aag?ttt?gga?ttc?agc?cta?cct?tat?gtt?cag?tat?ttt?gga?ggt 864
Met?Asp?Lys?Phe?Gly?Phe?Ser?Leu?Pro?Tyr?Val?Gln?Tyr?Phe?Gly?Gly
275 280 285
gtc?tct?gct?cta?agt?aaa?caa?cag?ttt?cta?acc?atc?aat?gga?ttt?cct 912
Val?Ser?Ala?Leu?Ser?Lys?Gln?Gln?Phe?Leu?Thr?Ile?Asn?Gly?Phe?Pro
290 295 300
aat?aat?tat?tgg?ggc?tgg?gga?gga?gaa?gat?gat?gac?att?ttt?aac?aga 960
Asn?Asn?Tyr?Trp?Gly?Trp?Gly?Gly?Glu?Asp?Asp?Asp?Ile?Phe?Asn?Arg
305 310 315 320
tta?gtt?ttt?aga?ggc?atg?tct?ata?tct?cgc?cca?aat?gct?gtg?gtc?ggg 1008
Leu?Val?Phe?Arg?Gly?Met?Ser?Ile?Ser?Arg?Pro?Asn?Ala?Val?Val?Gly
325 330 335
agg?tgt?cgc?atg?atc?cgc?cac?tca?aga?gac?aag?aaa?aat?gaa?ccc?aat 1056
Arg?Cys?Arg?Met?Ile?Arg?His?Ser?Arg?Asp?Lys?Lys?Asn?Glu?Pro?Asn
340 345 350
cct?cag?agg?ttt?gac?cga?att?gca?cac?aca?aag?gag?aca?atg?ctc?tct 1104
Pro?Gln?Arg?Phe?Asp?Arg?Ile?Ala?His?Thr?Lys?Glu?Thr?Met?Leu?Ser
355 360 365
gat?ggt?ttg?aac?tca?ctc?acc?tac?cag?gtg?ctg?gat?gta?cag?aga?tac 1152
Asp?Gly?Leu?Asn?Ser?Leu?Thr?Tyr?Gln?Val?Leu?Asp?Val?Gln?Arg?Tyr
370 375 380
cca?ttg?tat?acc?caa?atc?aca?gtg?gac?atc?ggg?aca?ccg?agc?tag 1197
Pro?Leu?Tyr?Thr?Gln?Ile?Thr?Val?Asp?Ile?Gly?Thr?Pro?Ser
385 390 395
<210>2
<211>398
<212>PRT
＜213〉mankind
<400>2
Met?Arg?Leu?Arg?Glu?Pro?Leu?Leu?Ser?Gly?Ser?Ala?Ala?Met?Pro?Gly
1 5 10 15
Ala?Ser?Leu?Gln?Arg?Ala?Cys?Arg?Leu?Leu?Val?Ala?Val?Cys?Ala?Leu
20 25 30
His?Leu?Gly?Val?Thr?Leu?Val?Tyr?Tyr?Leu?Ala?Gly?Arg?Asp?Leu?Ser
35 40 45
Arg?Leu?Pro?Gln?Leu?Val?Gly?Val?Ser?Thr?Pro?Leu?Gln?Gly?Gly?Ser
50 55 60
Asn?Ser?Ala?Ala?Ala?Ile?Gly?Gln?Ser?Ser?Gly?Glu?Leu?Arg?Thr?Gly
65 70 75 80
Gly?Ala?Arg?Pro?Pro?Pro?Pro?Leu?Gly?Ala?Ser?Ser?Gln?Pro?Arg?Pro
85 90 95
Gly?Gly?Asp?Ser?Ser?Pro?Val?Val?Asp?Ser?Gly?Pro?Gly?Pro?Ala?Ser
100 105 110
Asn?Leu?Thr?Ser?Val?Pro?Val?Pro?His?Thr?Thr?Ala?Leu?Ser?Leu?Pro
115 120 125
Ala?Cys?Pro?Glu?Glu?Ser?Pro?Leu?Leu?Val?Gly?Pro?Met?Leu?Ile?Glu
130 135 140
Phe?Asn?Met?Pro?Val?Asp?Leu?Glu?Leu?Val?Ala?Lys?Gln?Asn?Pro?Asn
145 150 155 160
Val?Lys?Met?Gly?Gly?Arg?Tyr?Ala?Pro?Arg?Asp?Cys?Val?Ser?Pro?His
165 170 175
Lys?Val?Ala?Ile?Ile?Ile?Pro?Phe?Arg?Asn?Arg?Gln?Glu?His?Leu?Lys
180 185 190
Tyr?Trp?Leu?Tyr?Tyr?Leu?His?Pro?Val?Leu?Gln?Arg?Gln?Gln?Leu?Asp
195 200 205
Tyr?Gly?Ile?Tyr?Val?Ile?Asn?Gln?Ala?Gly?Asp?Thr?Ile?Phe?Asn?Arg
210 215 220
Ala?Lys?Leu?Leu?Asn?Val?Gly?Phe?Gln?Glu?Ala?Leu?Lys?Asp?Tyr?Asp
225 230 235 240
Tyr?Thr?Cys?Phe?Val?Phe?Ser?Asp?Val?Asp?Leu?Ile?Pro?Met?Asn?Asp
245 250 255
His?Asn?Ala?Tyr?Arg?Cys?Phe?Ser?Gln?Pro?Arg?His?Ile?Ser?Val?Ala
260 265 270
Met?Asp?Lys?Phe?Gly?Phe?Ser?Leu?Pro?Tyr?Val?Gln?Tyr?Phe?Gly?Gly
275 280 285
Val?Ser?Ala?Leu?Ser?Lys?Gln?Gln?Phe?Leu?Thr?Ile?Asn?Gly?Phe?Pro
290 295 300
Asn?Asn?Tyr?Trp?Gly?Trp?Gly?Gly?Glu?Asp?Asp?Asp?Ile?Phe?Asn?Arg
305 310 315 320
Leu?Val?Phe?Arg?Gly?Met?Ser?Ile?Ser?Arg?Pro?Asn?Ala?Val?Val?Gly
325 330 335
Arg?Cys?Arg?Met?Ile?Arg?His?Ser?Arg?Asp?Lys?Lys?Asn?Glu?Pro?Asn
340 345 350
Pro?Gln?Arg?Phe?Asp?Arg?Ile?Ala?His?Thr?Lys?Glu?Thr?Met?Leu?Ser
355 360 365
Asp?Gly?Leu?Asn?Ser?Leu?Thr?Tyr?Gln?Val?Leu?Asp?Val?Gln?Arg?Tyr
370 375 380
Pro?Leu?Tyr?Thr?Gln?Ile?Thr?Val?Asp?Ile?Gly?Thr?Pro?Ser
385 390 395
Claims (8)
1. the application of β-4 galactosyltransferase I in preparation diagnosing cancer of liver preparation, wherein, with β-1,4 galactosyltransferase I activity in hepatic tissue or the blood sample as the diagnosing cancer of liver index; Described β-1,4 galactosyltransferase I increased activity is meant that β-1, the 4 galactosyltransferase I expression amount of tissue samples increases the perhaps increase of the quantity of β in the sample-1,4 semi-lactosi glycosidic bond.
2. application according to claim 1 is characterized in that, with β-1,4 galactosyltransferase I increased activity in hepatic tissue and the blood as the diagnosis index of liver cancer primary dcreening operation.
3. application according to claim 1 is characterized in that, with β-1,4 galactosyltransferase I increased activity in hepatic tissue and the blood as the diagnosis index of recurrence of PHC.
4. application according to claim 1, it is characterized in that, the β of hepatic tissue sample-1 wherein, the detection method of 4 galactosyltransferase I expression amounts is: the RNA that extracts the hepatic tissue sample, reverse transcription becomes cDNA then, the nucleotide coding sequence of amplification β-1,4 galactosyltransferase I, products therefrom increases more than the β that is the hepatic tissue sample-1, the 4 galactosyltransferase I expression amount of contrast.
5. application according to claim 1 is characterized in that, the detection method of β-1,4 galactoside bond number amount is in the described hepatic tissue sample: the hepatic tissue sample is made paraffin section, the dewaxing entry; Add H2O2 or methyl alcohol and block endogenic peroxidase, add avidin and block endogenic vitamin H; Sialidase digestion, sealing adds vitamin H-ricinus agglutinin-I overnight incubation then; Avidin-vitamin H that the adding mark is crossed is hatched, colour developing; Control group replaces vitamin H-ricinus agglutinin-I with sheep blood serum, perhaps adds the semi-lactosi competition and suppress when adding vitamin H-ricinus agglutinin-I.
6. application according to claim 5 is characterized in that, adopts horseradish peroxidase to carry out mark, the diaminobenzidine colour developing.
7. application according to claim 1, it is characterized in that, the detection method of β in the blood sample-1,4 galactoside bond number amount is: extract the serum protein in the blood sample, western blot transfers to pvdf membrane, sealing is spent the night, hatch with ricinus agglutinin-I that mark is crossed, wash film, detect then ricinus agglutinin-I in conjunction with quantity, the increase in conjunction with quantity of ricinus agglutinin-I is the increase of β in the hepatic tissue sample-1,4 galactoside bond number amount.
8. application according to claim 7 is characterized in that, adopts horseradish peroxidase to carry out mark, the ECL chemiluminescence system detect ricinus agglutinin-I in conjunction with quantity.
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| CN 200910197379 CN102041295B (en) | 2009-10-19 | 2009-10-19 | Application of beta-1,4 galactosy transferase I in preparing liver cancer diagnostic preparation |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105859807A (en) * | 2016-04-12 | 2016-08-17 | 成都大学 | Beta-1,4-galactosyltransferase I substrate fluorescent molecular probe, and intermediate and preparation method thereof |
| CN107884583A (en) * | 2016-09-30 | 2018-04-06 | 复旦大学 | Applications of the CD133 of cytoplasm positioning as prognosis in hcc mark |
| WO2020228721A1 (en) * | 2019-05-13 | 2020-11-19 | Lin Mei Chun | Method and kit for monitoring cancer |
| CN112067805A (en) * | 2020-08-25 | 2020-12-11 | 南通大学 | Application of beta-1, 4-galactosyltransferase-IsiRNA in drugs for overcoming drug resistance of liver cancer chemotherapy |
| CN113588951A (en) * | 2021-07-07 | 2021-11-02 | 武汉大学 | Application of ECA as molecular marker in preparation of kit for diagnosing and/or evaluating liver cancer in prognosis |
| CN113813257A (en) * | 2021-10-22 | 2021-12-21 | 吉林大学 | Application of biimidazole salt and drug-carrying system in serving as anticancer agent and anticancer preparation |
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2009
- 2009-10-19 CN CN 200910197379 patent/CN102041295B/en not_active Expired - Fee Related
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| 吴国强等: "β1―4半乳糖基转移酶受体底物的动力学 ", 《上海医科大学学报》 * |
| 江松敏，李溪冰，邵东敏，顾建新: "在大鼠诱发肝癌中β1，4半乳糖基转移酶的高效液相层析测定 ", 《上海医科大学学报》 * |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105859807A (en) * | 2016-04-12 | 2016-08-17 | 成都大学 | Beta-1,4-galactosyltransferase I substrate fluorescent molecular probe, and intermediate and preparation method thereof |
| CN107884583A (en) * | 2016-09-30 | 2018-04-06 | 复旦大学 | Applications of the CD133 of cytoplasm positioning as prognosis in hcc mark |
| WO2020228721A1 (en) * | 2019-05-13 | 2020-11-19 | Lin Mei Chun | Method and kit for monitoring cancer |
| CN112067805A (en) * | 2020-08-25 | 2020-12-11 | 南通大学 | Application of beta-1, 4-galactosyltransferase-IsiRNA in drugs for overcoming drug resistance of liver cancer chemotherapy |
| CN112067805B (en) * | 2020-08-25 | 2023-08-22 | 南通大学 | Application of beta-1, 4-galactosyltransferase-IsiRNA in medicines for overcoming drug resistance of liver cancer chemotherapy |
| CN113588951A (en) * | 2021-07-07 | 2021-11-02 | 武汉大学 | Application of ECA as molecular marker in preparation of kit for diagnosing and/or evaluating liver cancer in prognosis |
| CN113813257A (en) * | 2021-10-22 | 2021-12-21 | 吉林大学 | Application of biimidazole salt and drug-carrying system in serving as anticancer agent and anticancer preparation |
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|---|---|
| CN102041295B (en) | 2013-07-10 |
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