CN102631368B - Application of bone marrow mesenchymal stem cells (MSCs) to preparation of drugs for treating autoimmune diseases - Google Patents

Abstract

The invention discloses an application of bone marrow mesenchymal stem cells (MSCs) to preparation of drugs for treating autoimmune diseases. The bone marrow MSCs of the patients with autoimmune diseases have abnormity, which plays an important role in occurrence and development of the diseases. With unique immunoregulation and repair effects, the bone marrow MSCs can be applied to preparation of drugs for treating autoimmune diseases.

Description

The application of bone marrow interstital stem cell in preparation treatment autoimmune disease medicine

Technical field

The invention belongs to field of immunology, be specifically related to the application of bone marrow interstital stem cell in preparation treatment autoimmune disease medicine.

Background technology

1. bone marrow interstital stem cell function

Comprise two kinds of diverse stem cell in bone marrow: hematopoietic stem cell and interstital stem cell (mesenchymal stem cells, MSCs), the former maintains hemopoietic, and the latter has formed the microenvironment of bone marrow hematogenesis.These two kinds of independences and diverse stem cell not only coexist but also cooperate with each other on function, jointly complete into reproducing of human body inner blood system.Except bone marrow, also there is a small amount of MSCs in its hetero-organization, but in the adult in bone marrow content maximum, account for its nucleated cell 0.001～0.01%, so bone marrow is the MSCs source comparatively be applicable to.

The existence that Friedenstein in 1970 tests to have proved in vitro MSCs by fibroblastic colony units-forming; Through long-term research, people have had more deep understanding to its function.The adult stromal cell that bone marrow MSCs is pluripotency, can be divided into the tissue that multiple interstitial originates from, as skeletonization, cartilage and adipose cell; Under special environment, can also transdifferentiationof be multiple other histiocytes such as myocyte, neurocyte, vascular endothelial cell, liver pancreatic cell.MSCs has alternative repair to multiple damaged tissue, after implanting, can locate location and distribute as lung, bone, cartilage and skin etc. by the tissue beyond multiple hemopoietic.

Except differentiation, bone marrow MSCs also has stronger immunoregulation ability, and T, B, dendritic cell and NK cell are all had to the modulability inhibitory action.Research shows, but MSCs suppressor T cell propagation causes that the T cell differentiation stagnates, but and the suppressor T cell activation, lower the corresponding peptides section to naivety (naive) and Memorability antigen-specific T cytological effect, inducing T cell anergy (anergy); MSCs also can induce the generation regulatory T cells to be regulated and controled in addition.MSCs can suppress B cell proliferation, and (when B cell/MSCs is 1:1, effect is the strongest, and reach 1:5 or 1:10, time effect disappears), suppress the B cell differentiation and affect B cell chemotaxis (cultivate altogether rear B cellular expression Chemokines CC XCR4, CXCR5 and the minimizing of CXCR7 receptor with MSCs, thereby the chemotactic capability defect occurs).For dendritic cell, MSCs can suppress its propagation and differentiation and maturation, can stop mononuclear cell and bone marrow precursor to be divided into dendritic cell, suppresses CD1a, CD40, CD80, CD86 and HLA-DR and raises, and makes cell maintain crudity; MSCs also can change its cytokine secretion ability, thereby affects dendritic cell function.MSCs can suppress the propagation of NK cell equally, and its surface exists Multiple components can affect NK cell activation (comprise NKG2D part as MICA, ULBPs and DNAM-1 part as PVR, nectin-2).

2. interstital stem cell application present situation

MSCs can play supporting function by secrete cytokines in hematopoietic stem cell niche, and reduces PERIPHERAL BLOOD MONONUCLEAR CELL alloimmunity stimulating activity, thereby after promoting hematopoietic stem cell transplantation, hemopoietic system is rebuild.2000, the first term clinical trial showed, 28 routine women with breast cancer patients give the MSCs single infusion when accepting autologous hematopoietic stem cell transplantation, and its hemopoietic recovers to accelerate and without side reaction.Within 2005, another multicenter study also shows 46 routine patients capable allogeneic hematopoietic stem cell and the rear most hemopoietic recovery quickenings of MSCs treatment, not find the side effect simultaneously.

2004, Le Blanc reported first MSCs treatment graft versus host disease (GVHD) case, and infant is the Lymphocytic leukemia patient, MSCs is provided by its mother, infusion 1 time respectively when hematopoietic stem cell transplantation 70 days and 170 days, the infant doing well,improving, bilirubin level descends.2008, Le Blanc reported MSCs treatment serious difficult treated GVHD II phase clinical research result, treats altogether 55 routine patients, and 30 routine patients are alleviated fully as a result, another 9 routine partial rcsponses.The III phase is studied well afoot at present.

MSCs can repair cardiac muscle and vascular tissue simultaneously, have immunoregulatory activity, and there is easy cultivation amplification and meet the characteristics of transplanting needs, this makes it have theory value in treating cardiovascular disease, has been applied to the treatment of myocardial infarction, myocardial ischemia, heart failure at present.Through cardiac muscle, directly injecting is the most frequently used therapeutic modality, and preliminary study shows can improve cardiac ejection fraction, improves survival, but its efficacy and saferry still remains comparative study, is confirmed.

MSCs also has some Preliminary Applications at nervous system disease (as multiple sclerosis, amyotrophic lateral sclerosis), osteogenesis imperfecta, skin trauma.Due to the targeting of MSCs, have the report for oncotherapy in zooscopy.

3. autoimmune disease conventional therapy

Autoimmune disease (autoimlnune disease) refers to take the autoimmune response reaction class disease that to cause injuries of tissues and organs and corresponding function obstacle be main pathogenesis.Be a common clinically large class disease, putative autoimmune disease has kind more than 30 at least at present.According to autoimmune response, histoorgan is caused the scope of damage, usually autoimmune disease is divided the special and non-organ specificity two of organ large class.

Common autoimmune disease comprises clinically: connective tissue disease: rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA), systemic lupus erythematosus (sle) (SLE), sjogren syndrome (SS), polymyositis/dermatomyositis (PM/DM), scleroderma (SSC), systemic vasculitis; Neuromuscular disease: multiple sclerosis (MS), myasthenia gravis (MG), demyelination; Incretion disease: the virtuous upper gland cortical atrophy of constitutional, chronic thyroiditis, juvenile onset diabetes; Digestive system disease: chronic non-specific ulcerative colitis (Crohn disease), chronic active hepatitis, pernicious anemia and atrophic gastritis; Diseases of urinary system: autoimmune glomerulonephritis, lung nephrorrhagia syndrome; Disease in the blood system: autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura (ITP), idiopathic leukopenia.

The conventional treatments of autoimmune disease has:

(1) adrenocortical hormone: be the most frequently used medicines of most autoimmune diseasees, there is very fast Immunosuppression reaction and stronger antiinflammatory action.In recent years the Hormone stosstherapy adopted has improved the remission rate of critical illness, but hormone also has many side effect, prolonged application can suppress the hypothalamic-pituitary-adrenal system, the amount of antepituitary secretion thyroliberin obviously reduces, the corticoid secretion is reduced, atrophy appears in the cortex gland, thereby the needs of patients Long-term Hormone maintains treatment, and their stress ability weakens, flu and various infection easily appear, be subject to wound, cold wind stimulates, direct stimulation, during the stress state such as operative treatment Other diseases, hyposecretion due to hormone in vivo, may bring out on the one hand autoimmune disease occurs or increases the weight of, there will be on the other hand fatigue and weak, loss of appetite, hypoglycemic reaction, the symptom of the adrenal cortex secreting hormone deficiencies such as muscle arthralgia.In addition, hormone also can cause fat, hirsutism, hypertension, glaucoma, diabetes, digestive tract ulcer, hemorrhage, mental symptom, osteoporosis, femur head necrosis etc.

(2) immunosuppressant: autoimmune response has substantial connection to the genesis of most autoimmune diseasees, except hormone, in clinical position, toward contact, need apply several immunosuppressant of 1-to control the conditions of patients development.Immunosuppressant commonly used has cyclophosphamide, azathioprine, methotrexate, mycophenolate etc., and the major side effects of these medicines has gastrointestinal reaction, hematological, urinary system reaction, affects fertility, alopecia, increase Tumor incidence etc.Heavy dose of human immunoglobulin intravenous injection is by antibody sealing process treatment hormone and the out of contior intractable patient of immunosuppressant, successful experience is all arranged, but this method is very large to different patient's Different therapeutical effects, and certain toxic and side effects is also arranged both at home and abroad, expensive, apply limited.

(3) hematopoietic stem cell transplantation: although given hormone and immunosuppressant treatment, still have many patient's state of an illness to can not get controlling and maybe can not tolerate the serious adverse reaction that conventional therapy brings, this impels the clinical position, and the person constantly explores new Therapeutic Method.Hematopoietic stem cell transplantation (HSCT) is the treatment means grown up in the recent decade, mainly adopted autologous HSCT mode when the treatment autoimmune disease, its treatment principle has two aspects, the one, when pretreatment, destroy patient's abnormal immune system by high-dose chemotherapy or total irradiation, autoimmune disease is played to mitigation; The 2nd, the autologous peripheral blood stem cells of transplanting, to rebuild normal immune system, is blocked the generation of autoantibody or induces the immunologic tolerance to autoantigen in immunologic reconstitution.According to recent statistics, the whole world has nearly 1000 routine Intractable autoimmune disease patients to accept HSCT at present, comprising SSc 190 examples, and SLE 86 examples, PM/DM 14 examples, RA 86 examples, MS 368 examples, Crohn disease 21 examples.Owing to after the autologous HSCT of autoimmune disease, still having the some patients were state of an illness not control, some cases recurs after surgery, and the transplantation treatment risk is higher in addition, and related mortality is about 7%, higher than non-self immune disease (3%), restricted the extensive use of this technology.Transplant related mortality is relevant to concrete sick kind, is followed successively by from high to low SSc, SLE, MS and RA; Case selection, Supporting Therapy, pretreating scheme, whether go lymphocyte in graft etc. also to affect patient's survival rate.

Summary of the invention

The purpose of this invention is to provide the application of bone marrow interstital stem cell in preparation treatment autoimmune disease medicine.

Described autoimmune disease refers to a large class disease that comprises systemic lupus erythematosus (sle), systemic sclerosis, mixed connective tissue disease, rheumatoid arthritis, polymyositis/dermatomyositis, autoimmune hemolytic anemia, inflammatory bowel, Behcet disease, primary biliary cirrhosis etc.

In the present invention, described autoimmune disease refers in particular to systemic sclerosis, Crohn disease, polymyositis/dermatomyositis, hemolytic anemia, mixed connective tissue disease, rheumatoid arthritis, Behcet disease or primary biliary cirrhosis.

With following method, prepared by described bone marrow interstital stem cell:

(1) getting posterior superior iliac spine is point of puncture, with heparin diluent access puncture needle interface, slow bone marrow extraction, and dilutes with PBS;

(2) add the diluent through the described bone marrow of step (1) in lymphocyte separation medium (Ficoll), centrifugal after, the linen lymphocyte of sucking-off, then wash with PBS;

(3) at 37 ℃, 5% CO ₂, saturated humidity CO ₂in incubator, the lymphocyte that step (2) is obtained, cultivate with the L-DMEM complete culture solution containing 10% hyclone, changes first liquid after 24 h, removes non-adherent cell, within later every three days, with the L-DMEM complete culture solution, changes liquid.

(4) while being cultured to attached cell 90% fusion, gone down to posterity, used the PBS washed cell, used trypsinization, after the PBS washing, put 37 ℃, 5 %CO ₂, saturated humidity CO ₂incubator is cultivated with the L-DMEM complete culture solution in cultivating.

(5) get each and measure cell surface marker and measure its caryogram for bone marrow interstital stem cell, must there is the normal bone marrow interstital stem cell of interstital stem cell feature and karyotype.

The case that the inventor is a large amount of by investigation, find that autoimmune disease patient's bone marrow MSCs exists extremely, in the genesis of disease, plays an important role.For example the bone marrow MSCs growth in vitro calibration ordinary person of Patients with SLE is slow, and easily old and feeble, in the process that goes down to posterity (10 generation), vigor can be lost gradually, and interleukin-6,7mRNA express reduction, and MSCs exists cytoskeleton abnormal.The nearest research of inventor finds that again the external compared with normal of the differentiation capability to fat and chondrocyte of systemic lupus erythematosus (sle) bone marrow MSCs obviously reduces, and is transplanted to skeletonization in nude mouse and also obviously descends.In addition, while also finding that the bone marrow MSCs of Patients with SLE and PBMC of healthy people are cultivated in vitro altogether, the ability that stimulates regulatory T cells (Treg) to raise also is starkly lower than normal MSCs.Because the autoimmune disease patient exists MSCs abnormal, therefore, autoimmune disease is not only " hematopoietic stem cell disease ", and is " interstital stem cell disease ".This is also why autologous HSCT treats the high reason of autoimmune disease relapse rate, and high rejection, high mortality and high cost that allosome HSCT exists have limited its clinical practice.Because bone marrow MSCs has unique immunomodulating and repair, so can be applied in preparation treatment autoimmune disease medicine.

The present invention proposes the application of bone marrow MSCs in the medicine of preparation treatment autoimmune disease.The medicine of the treatment autoimmune disease that adopts bone marrow MSCs to prepare is compared with autoimmune disease conventional therapy medicine, have the following advantages: bone marrow MSCs is as the active component for the treatment of autoimmune disease medicine, unique immunomodulating and repair are arranged, be used for the treatment of autoimmune disease and there is " treatment convenience, curative effect is lasting " characteristics, can help the patient to reduce the various side effect that the kind of Drug therapy and quantity and medicine are brought, the Drug therapys such as the immunosuppressant of even may stopping using, reduce mortality rate and disability rate, improve patients ' life quality, simultaneously, the medicine of the treatment autoimmune disease that the bone marrow MSCs of take the is active component biological preparation expense novel than other is low, and most of patients has corresponding economic capability.Therefore the application of bone marrow MSCs in preparation treatment autoimmune disease medicine has huge society and economic benefit.

The accompanying drawing explanation

Fig. 1 and Fig. 2 mean that cd patient bone marrow MSCs transplanting ideocolon mirror changes, and wherein Fig. 1 means to transplant front sieve grace patient intestinal mirror photo, and Fig. 2 means to transplant rear first quarter moon sieve grace patient intestinal mirror photo;

Fig. 3 and Fig. 4 mean that patient with dermatomyositis bone marrow MSCs treatment front and back muscle nuclear magnetic resonance, NMR (MRI) change, and wherein Fig. 3 means the front patient with dermatomyositis muscle MRI of bone marrow MSCs treatment, and Fig. 4 means the rear patient with dermatomyositis muscle MRI of bone marrow MSCs treatment.

The specific embodiment

Embodiment 1 bone marrow interstital stem cell in-vitro separation, cultivation

(1) source of cell

The MSCs that is used for infusion of therapeutic autoimmune disease patient derives from the allosome BMNC and cultivates and form through amplification in vitro.

(2) collection of cell

1 device therefor, material, reagent

Superclean bench, laminar flow cleaning bed, heparin, injection normal saline, disposable empty needle and bone marrow aspiration bag.

The acquisition method of 2 bone marrow

(1) disinfection by ultraviolet light superclean bench 30 min.

(2) configuration heparin-normal saline in super-clean bench: the heparin that adds 65.5 U with 1 ml normal saline.

(3) the Marrow donation person is prostrate on the laminar flow cleaning bed, and getting posterior superior iliac spine is point of puncture, routine disinfection paving hole towel, with the marrow puncture needle puncture, after fixing, the 20ml injection needle is taken out heparin diluent 5ml, access puncture needle interface, slowly take out bone marrow to 20ml scale place, extracts altogether 2 empty needles.

(3) bone marrow MSCs is separated and is cultivated

1 device therefor, material and reagent

Superclean bench, CO ₂incubator, inverted microscope, horizontal type centrifuger, constant water bath box, high-pressure steam sterilizing pot, Tissue Culture Dish (f10 cm), centrifuge tube (50ml) and suction pipe (5ml, 10ml).

Lymphocyte separation medium (Ficoll) L-DMEM culture fluid, peptide Ox blood serum, PBS liquid and pancreatin.

The primary separation of 2 bone marrow MSCs, cultivation

(1) get 2 of 50ml centrifuge tubes, every pipe adds bone marrow fluid 20 ml, stand-by with one times of PBS dilution;

(2) get 4 of 50 ml centrifuge tubes, every pipe adds 20 ml Ficoll lymphocyte separation mediums (proportion 1.077);

(3) slowly in lymphocyte separation medium (Ficoll), add the bone marrow fluid after dilution along tube wall, every pipe adds dilution bone marrow fluid 20ml;

(4) by gained mixture in step 3 centrifugal (room temperature 2000 rpm, 30min);

(5) with the middle buffy coat of suction pipe inserting step (4) gained sample, the linen lymphocyte of sucking-off, to another centrifuge tube, adds PBS to 40 ml gently, mixes, and after centrifugal (room temperature 1000 rpm, 5min), abandons supernatant, and the PBS repeated washing once;

(6) the L-DMEM complete culture solution for cell (containing 10% hyclone) separated being adjusted to cell concentration is 5 * 10 ⁶individual cell/ml, then be inoculated in culture dish, puts 37 ℃, 5% CO ₂, saturated humidity CO ₂incubator is cultivated;

Change first liquid after (7) 24 h, remove non-adherent cell, later every 3d changes liquid with the L-DMEM complete culture solution.

3 bone marrow MSCs go down to posterity

(1), while being cultured to attached cell 90% fusion, gone down to posterity;

(2) absorb old culture fluid in culture dish, add PBS, the wave and culture ware, to wash away the culture fluid in remaining in, absorb PBS gently;

(3) add pancreatin 2 ml in culture dish, the wave and culture ware, make Digestive system flow through all cells surface gently; Observe under inverted microscope, find the Cytoplasm retraction, after intercellular substance increases, should add immediately complete culture solution 2ml to stop digestion;

(4) repeatedly blow and beat the bottle parietal cell, after cell detachment bottle wall, form cell suspension;

(5) cell suspension is sucked to centrifuge tube, centrifugal (room temperature 1000 rpm, 5min), abandon supernatant, and after the PBS dilution, supernatant is abandoned in washing;

(6) adjusting cell concentration with the L-DMEM complete culture solution is 5 * 10 ⁶individual cell/ml, then be inoculated in culture dish, every bottle of 8 ml; Put 37 ℃, 5 %CO ₂, saturated humidity CO ₂incubator is cultivated.

(7) while reaching the third generation (P3), collecting cell is standby.

4 bone marrow MSCs cell cryopreservations

(1) digestion attached cell, (1)-(5) in step same 3;

(2) add 0.1ml dimethyl sulfoxide (DMSO), the 0.9ml hyclone, move into cell cryopreservation tube;

(3) freeze-stored cell (4 ℃, 1 hour;-20 ℃, 4 hours;-70 ℃, 12 hours, liquid nitrogen container was preserved).

5 bone marrow MSCs cell recoveries

(1) take out cryopreservation tube, put into immediately 37 ℃ of water-baths, shake, thaw rapidly in 1 min;

(2) suction pipe sucking-off cell suspension, put into 50 ml centrifuge tubes, adds 40ml PBS, piping and druming;

(3) centrifugal (room temperature 1000 rpm, 5min), abandon supernatant, repeats once;

(4) add the L-DMEM complete culture solution to be inoculated in culture dish,

(4) evaluation of bone marrow MSCs

1 total cellular score

The cumulative volume of measurement definite cell suspension.After fully mixing, draw 0.1ml cell suspension and drip on cell counting count board, counting.Calculate total cellular score.

2 living cells ratios

Carry out Trypan Blue, counting gathers living cells proportion in cell.

3 flow cytometers detect cell surface marker

Get each for CD14, CD29, CD34, CD44, CD45, the CD105 effect of bone marrow MSCs with fluorescein isothiocyanate (FITC) (Chinese name, full name) or phycoerythrin (PE) labelling, the flow cytometer detection.Result demonstration CD14, CD14 and CD45 are negative, and CD29, CD44 and CD105 are positive, are rendered as the feature of interstital stem cell.

4 bone marrow MSCs karyotypes are measured

Get each for bone marrow MSCs, in culture supernatant, adding Colchicine to make its ultimate density is 1 μ g/ ml, continues to cultivate 30min, abandon supernatant, 1% sodium citrate hypotonic medium is processed 50min, with fixative (methanol: glacial acetic acid 3:1) fixing, the original position harvesting, 45 ℃ of roasting sheets spend the night, the aobvious band of trypsin process G.15 metacinesis phases of every routine specimen counting, analyze 5 caryogram.This karyotype testing result shows normal dyeing body caryogram.

The medication of embodiment 2 bone marrow MSCs

(1) bone marrow MSCs final evaluation before using

1 sterility test

As any abnormal in do not occurred in incubation, routine first 3 days bone marrow MSCs infusion patient, after changing liquid for the last time, make sterility test to the supernatant in each culture bottle, culture of bacteria and mycete.Sterility test is undertaken by the regulation of " Products in China rules (1995) ".Cell conditioned medium liquid must not have antibacterial, fungus growth.

2 outward appearances and Microscopic observation

On the same day of infusion, without antibacterial, fungus growth, the supernatant in culture bottle is carried out to color observation as sterility test report, under inverted microscope observation of cell form, cell density, have or not antibacterial or fungus growth.If any suspicious, must again sample and do sterility test culture of bacteria, mycete.

3 living cells ratios

Infusion accounted for more than 95% the cell suspension sampling check living cells ratio of collecting the same day.

4 karyotypes

First 3 days sampling row karyotypes of bone marrow MSCs infusion inspection: should be normal dyeing body caryogram.

5 surface markers

First 3 days sampling row surface markers of bone marrow MSCs infusion are measured: CD14, CD34, CD45 express negative; CD29, CD44, CD45 express positive.

(2) medication of bone marrow MSCs

1 device therefor, material, reagent

Superclean bench centrifuge disposable syringe injection normal saline human albumin

Operation before 2 infusions

When the total cellular score amount reaches (1～2) * 10 ⁶/ kg body weight, can consider the infusion preparation:

before infusion, sampling is left and taken cell culture supernatant and is made antibacterial, fungal culture, detection of mycoplasma and endotoxin measurement;

before infusion, cell is got in sampling, and counting cells concentration, living cells ratio, cell surface marker, cell chromosome caryogram are measured, and specifically see the evaluation of bone marrow MSCs.

3 infusion operations

trypsinization all cells (operation is the same), centrifugal (room temperature 1000 rpm, 5min), abandon supernatant, repeatedly fully washing; Add the normal saline 100ml containing 5% human albumin, the cell culture fluid of residual less than 0.5% only in the cell suspension of infusion.Extract 0.1ml cell suspension counting.Indicate the patient's name, prepare infusion.

before cell infusion, half an hour is to patient's vein prednisolone 40mg.

open hand back vein, input a small amount of normal saline with the blood exchange transfusion set, and the separation bag of cell is equipped with in unobstructed rear connection, adjusts infusion velocity, observes patient's reaction in infusion process, in time anti symptom treatment.Approximately 15 minutes consuming time of whole process.

The application of embodiment 3 bone marrow MSCs in the medicine of the different autoimmune diseasees of preparation treatment

(1) application of bone marrow MSCs in the medicine for preparing the transplantation in treating systemic sclerosis

1. case inclusion criteria: 1) be diagnosed as systemic sclerosis, 2) used hormone and immunosuppressant treatment more than half a year more than a kind, without obvious curative effects person, 3) the signature Informed Consent Form agrees the MSCs treatment.

2. exclusion standard: 1) patient exists serious infection, 2) disease whole latter stage, occurred that the important organ function is badly damaged.

3. transplantation Project: the input of bone marrow MSCs vein, cell number is 1 * 10 ⁶/ kg body weight.

4. treatment is estimated: treat altogether 6 routine patients, wherein male's 4 examples.Before transplanting, transplant after January, March, December patient's visceral injury and skin sclerosis are scored and are assessed, result shows that patient's heart, lung, renal function keep stable, the skin integration progressively improves, the skin ulcer healing is accelerated.

5. model case:

Wang * *, the female, 45 years old, because of " both hands refer to swelling, stiff 7 years companions are uncomfortable in chest, the half a year of panting ", (2008-01-15) was admitted to hospital.The patient before 7 years without under obvious inducement, occur both hands meet coldly turn white, blue phenomenon, feel and the both hands numbness gradually occur that the swelling of symmetry finger is stiff, finger skin is tightened sense, joint deadlock in morning, pain between companion's finger, symptom increases the weight of gradually, in the court diagnosis " systemic sclerosis ", gives prednisone, Ai Ruohua is oral always, cyclophosphamide 0.4g intravenous drip, more than first quarter moon once, the mistake that fades of both hands interphalangeal joint pain, symptom gets nowhere.Occur nearly half a year by " mask face ", before half a year, began to occur without nervous under inducement, and uncomfortable in chest after the appearance activity, asthma, put down the sleeping dyspnea of feeling night, two lower limb, ankle place edema gradually appear, the discomforts such as companion's abdominal distention, poor appetite, the meeting section that haves a medical check-up, the visible a small amount of telangiectasis of neck skin, edema of pair of lower extremities, the visible skin diabrosis of left lower extremity, both hands refer to swelling, the finger tip cyanosis, and skin elasticity goes down, clench fist limited, antinuclear antibody granular pattern strong positive is shown in auxiliary inspection, the anti-RNP positive, Color Sonography prompting pulmonary hypertension 87mmHg.Successively promoting the circulation of blood of patient slurry displacement three times and combine hormone, CTX treat unsatisfactory curative effect, in the capable allogenic bone marrow mesenchymal stem cell transplantation of 2008-01-23 art (donor is the patient husband), common transfusion cell several 6 * 10 ⁷, the deliquescing of postoperative patient skin, the diabrosis of left lower extremity skin before shoals.

(2) application of bone marrow MSCs in the medicine of preparation treatment Crohn disease

1. case inclusion criteria: 1) be diagnosed as Crohn disease, 2) more than having adopted conventional method (hormone and immunosuppressant more than a kind) treatment half a year, unsatisfactory curative effect person, 3) the signature Informed Consent Form agrees the MSC treatment.

2. exclusion standard: the patient of complicated with severe infections.

3. transplantation Project: the input of bone marrow MSCs vein, cell number is 1 * 10 ⁶/ kg body weight.

4. treatment is estimated: treat altogether 3 routine patients, be the male.Before transplanting, after transplanting, January, March, December are assessed patient's symptom, and are carried out enteroscopy.Result shows that patient's stomachache, symptom of diarrhea alleviate, intestinal section ulcer healing, and a routine patients with intestinal fistula secretions obviously reduces.

5. model case:

Old *, the male, 27 years old, because of " two hip pain 12 years, repeatedly separate magma just half a year ", (2008-04-17) was admitted to hospital.Lumbosacral region appears in the obvious inducement of patient more than ten Nian Qianwu, coxa joint pain, more serious during static rest, after activity, slightly be improved, rear disease progression, arthralgia increases the weight of, and there is ophthalmia to show effect, the sacroiliac joint x-ray shows slight sacroiliitis, be diagnosed as " ankylosing spondylitis ", give " methotrexate, Ying Taiqing, Medrol, sulfasalazine, benefit match is general " etc. treatment, recurrent diarrhea appears in October, 2007, stool is the magma sample, 3-7 times/day, in amount, companion's hypogastric region feeling of repletion, alleviate after an action of the bowels, intestinal mirror prompting multiple ulcers, rotten to the corn, diagnosis " Crohn disease ".In capable allogenic bone marrow mesenchymal stem cell transplantation art on April 24th, 2008 (donor is mother patient), transfusion cell several 6.5 * 10 altogether ⁷, postoperative without abdominal pain diarrhea, check electronics intestinal mirror shows that transverse colon to terminal ileum place has many pieces of polyps to form, and polyp size does not wait, and the part polyp shows to fester, and before being admitted to hospital, takes an evident turn for the better.The patient left hospital on April 30th, 2008.After leaving hospital, one week patient is without the abdominal pain diarrhea symptom, and check electronics intestinal mirror shows the slight polypoid proliferation of terminal ileum, and descending colon subregion interstitial edema, have no other abnormal (as Fig. 1 and Fig. 2).

(3) application of bone marrow MSCs in the medicine of preparation treatment polymyositis/dermatomyositis

1. case inclusion criteria: 1) be diagnosed as polymyositis/dermatomyositis, 2) used hormone and immunosuppressant treatment more than half a year more than a kind, without obvious curative effects person, 3) the signature Informed Consent Form agrees the bone marrow MSCs treatment.

2. exclusion standard: 1) patient exists serious infection, 2) disease whole latter stage, occurred that the important organ function is badly damaged.

3. transplantation Project: the input of bone marrow MSCs vein, cell number is 1 * 10 ⁶/ kg body weight.

4. treatment is estimated: treat altogether 3 routine patients, wherein male's 2 examples.Before transplanting, after transplanting, January, March, December are assessed patient's muscular strength, skin lesion situation, and parallel creatase, muscle MRI check that the judgement muscle inflammation has unchanged.After result shows patient treatment, the myasthenia symptom is improved, and creatase descends rapidly and keeps stable.

5. model case:

Zhu * *, the man, 21 years old, because of " limbs fatigue companion erythra is more than 1 year repeatedly, increases the weight of one week ", (2008-06-23) was admitted to hospital.Electrocardio diagram tachycardia, myocardial enzymes CK-MB41U/L, CK496U/L, but negative troponin, appear that thereafter cardiopalmus is uncomfortable in chest, in patient's rear sense nasal obstruction watery nasal discharge of suffering from cold before more than a year.Two lower limbs edema, weak occur subsequently, look into CK-MB 74U/L, CK 2372U/L, ultrasoundcardiogram is roughly normal, and euthyroidism gives the treatments such as diuresis, cardiac nutrition and takes a turn for the better.Forehead appears the year before, cheeks edema erythema, erythra is lamellar, exceed surface, without scratching where it itches pain, between erythra, skin is rubescent, erythra is without ulceration, electromyogram prompting muscle-derived changes, be diagnosed as " dermatomyositis ", repeatedly occur that over one year creatase raises, be up to the 4000u/L left and right, once used " MMF except hormone, cyclophosphamide impacts, plasmapheresis " etc. treatment, effect is not remarkable, before one week after the sense activity limbs fatigue increase the weight of, right leg is ached obviously, have a medical check-up after being admitted to hospital 2 grades of right lower extremity muscular strengths, muscular tenderness (+), 3 grades of left lower extremity muscular strengths, muscular tension is normal, upper limb muscular strength 4 ^-level, auxiliary looking into shown ALT 241.9U/L, AST 171.8U/L, GGT 475.2U/L, LDH 1032U/L, CK 2007U/L, CK-MB 156U/L, α-HBDH 828.9U/L(2008-06-25).

In capable allogenic bone marrow mesenchymal stem cell transplantation art on July 4th, 2008 (father patient is for marrow), the transfusion cell number is 8 * 10 altogether ⁷, in art, without uncomfortable, in postoperative January, MRI shows that muscle changes recovers normally that (as Fig. 3 and Fig. 4, the front muscle inflammation for the treatment of is obvious, as shown by arrows) substantially.The situation of following up a case by regular visits to is as following table.This patient can normally go to school at present.

Annotate: NA means not detect

(4) application of bone marrow MSCs in the medicine of preparation treatment hemolytic anemia

1. case inclusion criteria: 1) be diagnosed as autoimmune hemolytic anemia, 2) used hormone and immunosuppressant treatment more than half a year more than a kind, without obvious curative effects person, 3) the signature Informed Consent Form agrees the MSCs treatment.

2. exclusion standard: 1) patient exists serious infection, 2) disease whole latter stage, occurred that the important organ function is badly damaged.

3. transplantation Project: the input of bone marrow MSCs vein, cell number is 1 * 10 ⁶/ kg body weight.

4. treatment is estimated: treat altogether 2 routine patients, be the women.Before transplanting, after transplanting, January, March, December are assessed patient's symptom and hemoglobin level.Result shows the patient treatment doing well,improving, and hemoglobin is gone up rapidly.

5. model case:

Yin * *, the female, 43 years old, because of " dizziness, malaise more than 2 years ", to be admitted to hospital (on March 20th, 2008), the auxiliary routine of having a blood test is shown WBC 12.4 * 10 ⁹/ L, RBC 1.6 * 10 ⁹/ L, Hb 56g/L, PLT 362 * 10 ⁹/ L, reticulocyte 22.66%; The routine urinalysis trace of occulting blood, urine bilirubin 1+, be diagnosed as autoimmune hemolytic anemia (AIHI).Successively give the treatments such as cyclophosphamide, Mabthera, ciclosporin A, hormone maintains 30～40mg/ day always, therapeutic effect is not obvious, Hb is gradual decline, (08-03-26) 47g/L of 48g/l → (08-03-27), give the treatments such as MMF, prednisolone after Hb rise to some extent, (08-04-08) 71g/L of 69g/L → (08-04-28), viral infection appears in rear patient, aggravation, 2008-05-13:Hb48g/L.

In on 05 08th, 2008 capable allogenic bone marrow mesenchymal stem cell transplantation (son patient is for marrow), transfusion cell several 7.2 * 10 altogether ⁷, after transplanting, 3d check routine blood test shows Hb 92g/L, and routine urinalysis is normal, and the prednisolone decrement is 30mg/ day, the MMF of stopping using.After transplanting, one week check routine blood test shows Hb 96g/L, is left hospital, and continues to take prednisone 30mg/ day, CTX0.4g/ first quarter moon seance.

(5) application of bone marrow MSCs in the medicine of preparation treatment mixed connective tissue disease

Jiang * *, the female, 27 years old, because " heating companion muscle arthralgia is 4 years repeatedly, ecchymosis March " (2007-07-02) is admitted to hospital, the patient low grade fever occurred without obvious inducement before 4 years, companion's whole-body muscular pain, extremity size joint migration mild pain, hand swelling, both hands are met cold rear blue turning white, the cervical lymph node enlargement, tenderness is arranged, after this low grade fever appears in patient repeatedly, companion's muscle arthralgia, the cervical lymph node enlargement is arranged, auxiliary inspection ANA granular pattern (+), anti-RNP(+), pulmonary function shows that CO disperse value is normal 63%, anti-Sm(-), examine as " mixed connective tissue disease ", always with " Medrol 12mg bid, cyclophosphamide 0.4g/ first quarter moon " treatment, before March, the patient ecchymosis occurs without the inducement extremity, with lower limb, attach most importance to, without oral cavity, nasal bleeding, menorrhagia, the auxiliary PLT 29 * 10 that looks into ⁹/ L.Give the treatments such as prednisolone, oxychloroquine, danazol, ciclosporin A, vitamin E, Gaierqi D (vitamin D3 and calcium carbonate) after being admitted to hospital, be interrupted and adjust prednisolone dosage, be interrupted and use " vincristine " totally three times during treatment, 2007-07-13 ~ 07-15 gives the third ball 15g and impacts 3 days, 08-27 to 08-28 gives prednisolone 1g and impacts 2 days, and 08-29 to 09-02 again gives the third ball 20g and impacts 5 days platelet still without (9-11) * 10 of obviously rising ⁹/ L, think that through expert consultation this patient has the splenectomy indication, in 2007-09-06, proceeds to the parallel splenectomy of department of general surgery, and the postoperative patient platelet fluctuates in (40-50) * 10 ⁹/ L, peritoneal cavity drainage tube is in place unobstructed, in 2007-09-10, goes back to my section, and within postoperative two weeks, check PlT shows 101 * 10 ⁹/ L.The capable allogenic bone marrow mesenchymal stem cell transplantation of 2007-09-27 art (father patient is for marrow), transfusion cell several 6.5 * 10 altogether ⁷, postoperative patient is without discomfort, and PlT maintains normal level always, in 2007-10-03, leaves hospital.

(6) application of bone marrow MSCs in the medicine of preparation treatment rheumatoid arthritis

Old * *, the female, 38 years old, because of " extremities joint swells and ache stiff 3 years of morning of companion, increases the weight of one day ", (2008-10-01) was admitted to hospital.The patient before 3 years without inducement occur both hands refer between, metacarpophalangeal joints pain, companion morning deadlock, start to occur that both shoulders, elbow, wrist, knee joint, the palm refer to, interphalangeal joint swells and ache in November, 06, companion morning deadlock>1h.Outer court looks into breast CT and shows that two interstitial lungs sexually revise, anti-CCP antibody positive, and RF189 IU/L, be diagnosed as " rheumatoid arthritis, interstitial pneumonia ", and the treatment such as prednisone, methotrexate is alleviated.Last Dec, membra arthralgia increased the weight of again, and 3 symptoms of promoting the circulation of blood slurry displacement are obviously alleviated, and continue to use Medrol, oxychloroquine, sulfasalazine, cyclophosphamide, and the whole body arthralgia still has repeatedly.Before 1 day the previous day difficulty in walking the whole body arthralgia appearred, after inactive hormone voluntarily.Health check-up is shown in that two wrists, double knee joint, the both hands palm refer to, interphalangeal joint swelling, and tenderness is obvious, limitation of activity, and auxiliary inspection ESR 64mm/h, the DAS28 scoring is 8.58.Patient's rear relevant auxiliary inspection of improving of being admitted to hospital, pretreatment gives CTX0.8 * 4d, in the capable allogene mesenchymal stem cell transplantation of 2008-10-08 art (the patient husband is for marrow), transfusion cell several 7 * 10 altogether ⁷, postoperative without uncomfortable in art.After transplanting, two weeks patient's joint swelling and pains are clearly better, and the DAS28 scoring is 4.54, ESR27 mm/h, and improvement is left hospital.

(7) application of bone marrow MSCs in the medicine of preparation treatment Behcet disease

?hao * *, the female, 50 years old, because of " oral ulcer is 5 years repeatedly; arthralgia 2 years ", (2008-07-11) was admitted to hospital, be diagnosed as " Behcet disease ", successively take " reaction stops, likes Novartis, Theragran, colchicine ", vein is used in conjunction 4 months with cyclophosphamide 0.4g/ first quarter moon, arthralgia takes a turn for the better, but the oral ulcer sustainable existence, stem cell transplantation between the capable allogene of 2008-07-16, transfusion cell several 6 * 10 altogether ⁷, the patient is without discomfort, and the oral ulcer symptom takes a turn for the better, and 2008-07-19 leaves hospital.

(8) application of bone marrow MSCs in the medicine of preparation treatment primary biliary cirrhosis

Liu * *, the female, 38 years old, because of " skin itching repeatedly; yellow sclera August, xerostomia, weak March " be admitted to hospital (2008-08-08), auxiliary looking into shown AST 100.9U/L, ALT 112.0U/L, AKP 312.6U/L, GGT 267.6U/L, TBil 32.6umol/L, DBil 17.0umol/L, TG 1.71mmol/l, ANA positive (nuclear membrane type), the mitochondrial antibody M2 positive, four HA 555.66ng/ml of hepatic fibrosis, PCIII1 88.84ug/l, IVC 97.34ng/ml, LN 79.18ng/ml.Be diagnosed as " primary biliary cirrhosis ", in the capable allogenic bone marrow mesenchymal stem cell transplantation of 2008-08-12 art (the patient husband is for marrow), transfusion cell several 6.5 * 10 altogether ⁷, postoperative without uncomfortable, check liver function: AST7 9.2U/L, ALT 95.6U/L, AKP 300.3U/L, GGT 299.1U/L, TBil 30.5umol/L, DBil 17.4umol/L.

2008-10-08(transplants latter two month) check liver function: ALT 113 U/L, AST 106 U/L, ALP 284 U/L, GGT 228 U/L, TBil 29.8 umol/L, DBil 12.5 umol/L, TP 29 mmol/L.

The case that the inventor is a large amount of by investigation, find that autoimmune disease patient's bone marrow MSCs exists extremely, in the genesis of disease, plays an important role.Because MSCs has unique immunomodulating and repair, so can be applied in preparation treatment autoimmune disease medicine, not only comprise the application in the medicine of above-mentioned systemic sclerosis, Crohn disease, polymyositis and dermatomyositis, hemolytic anemia, mixed connective tissue disease, rheumatoid arthritis, Behcet disease or the primary biliary cirrhosis of mentioning in preparation treatment embodiment, also be included in the application in other autoimmune disease of preparation treatment.

Claims (1)

1. the application of bone marrow interstital stem cell in preparation treatment polymyositis and dermatomyositis medicine.

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