The Repair Effect Of Induced Bone Marrow Mesenchymal Stem Cells On The Islet Function Of Diabetes Mellitus

Part one Study of induced differentiation of bone marrow mesenchymal stem cells by pancreatic stem cellsObjective: Stem cells are the initial source of body and various tissue cells and their most significant biology characteristic is the ability of self-renewal,proliferation and the multi-directional differentiation potential.According to its different sources,stem cells can be divided into embryonic stem cells cells,induced pluripotent stem cells and adult stem cells.Bone mesenchymal stem cells(BMSC)is the other type of stem cells apart from hematopoietic stem cells in bone marrow,rendering fibroblasts form in vitro culture.In recent years,it was found that BMSC can differentiate into many cell types,such as cartilage cells,fat cells,myocardial cells and endothelial cells of mesoderm,the ectoderm neurons,hepatic oval cells of endoderm.However,more and more experimental evidences show that BMSC also has a role on immune regulation,anti-inflammatory,promoting angiogenesis and nutrition.Further more compared with embryonic stem cells or other tissue specific stem cells,mesenchymal stem cells have many advantages,such as easy to get,fewer ethical limits and low immunogenicity.This makes BMSC to be an ideal seed cells in tissue engineering,regenerative medicine and autoimmune disease treatment.However,directly transplant BMSC can often not reach the ideal therapeutic effect,because of the lack of target for treatment.Pancreatic stem cells(PSCS)are a kind of adult stem cells existing in fetal and adult pancreas,which maintaining self-renewal and multiple differentiation potentials.However,because of the lack of pancreatic stem cell,immune rejection and so on,the application of PSCS is liminted.In addition,neither direct transplantation or transplantation of induced islet like cell mass, can achieve the desired therapeutic effect.In this study,we use the co-culture method of pancreatic stem cells(PSCS)and bone marrow mesenchymal stem cells(BMSC)to induce the differentiation of BMSC into pancreatic stem like cells,thus providing the basis for its application in diabetes.The morphology of primary cultured BMSC and PSCS cells were observed by light microscope.Flow cytometry and immunofluorescence were used to identify BMSC and PSCS.RT-PCR and immunofluorescence were used to detect the differentiation marker genes of induced BMSC,in order to investigate the effect of PSCS on the differentiation of BMSC.Methods: 1 Isolation and culture of primary bone marrow mesenchymal stem cells The whole bone marrow adherence method was used.After anesthesia and execute SD rats(about 80g),the femur was taken out and placed in a flat plate with PBS,to wash 3 times.Cut off the proximal femoral epiphysis,and repeatedly wash the bone marrow cavity until the femur was pale with a 5 m L syringe draw 5 % FBS-MSCM medium from the distal epiphysis of holes into the needle.Gently percuss the bone marrow fluid and centrifugal 5 min at room temperature for 800 rpm.Washed 2 times with PBS,the cells were inoculated in 25 cm2 culture bottle with 2~3 m L medium.After the extraction of 24 h,wash two times with the PBS when change the liquid to remove the mixed cells,and passage for first time when the density of cells was about 80 % in 5~7 d.Then each time about 2~3 d can be passaged.Morphological changes of cells were observed under light microscope.2 Isolation and culture of primary pancreatic stem cells Collagenase digestion combined differential adherence method was used.After anesthesia and execute SD fetal rat,the pancreatic buds were taken out and put into the penicillin glass bottle with a pre cooled PBS,and then washed with PBS for 3 times.With preheating 500 μL 0.5 mg/m L collagenase V,gently shake the glass bottle 10 min in 37℃water bath,repeating and collecting the supernatant until the organization into sandy.Add 5 % FBS-hanks solution in the collection of the supernatant to terminate the digestion and gently triturate the liquid into cell suspension.The cell suspension was centrifuged 5 min at room temperature for 800 rpm,and precipitated two times with Hanks,then repeated centrifugation after washing 3 times.Finally the precipitation gently inoculate in 25 cm2 flask with 3 m L 5 % FBS-1640 medium and culture in 37℃。After extraction of every 1 h,put the cell suspension into another bottle.Collect the cells differential adherencing for 3 or 4 times,and then culture.After 2~3 d,the cells shows cobblestone like and then continuing to culture 1~2 d,the cells can be passaged when density reached 80 %.Morphological changes of cells were observed under light microscope.3 Identification of surface markers of bone marrow mesenchymal stem cells by flow cytometry Collect the P3 generation of cells,digest with trypsin,centrifugal 5min for 1000 rpm,and wash the cells with PBS for 3 times.After counting cells,CD105,CD90,CD44,CD34 and CD45 antibodies were added into each tube,and the same type of control was established in each tube.The tubes incubate protecting form light at room temperature for 40 min and wash 3 times with PBS to remove the unbound antibody,and then add 500 μL PBS to the cell suspension.Analysis in flow cytometry.4 Identification of pancreatic stem cells by immunofluorescence Inoculate the good condition P2 generation cells in pre-placed sterilized glasses 6-well plates,and slide out the glasses for immunofluorescence staining of Nestin and Ngn3 to detect their distribution and expression,after growing 24 h to fusing to 80 %.5 Co-culture of pancreatic stem cells and bone marrow mesenchymal stem cells Cells were divided into BMSC group,PSCS group and CC group.6 Detect the expression of nestin and ngn3 m RNA by RT-PCR method Using Trizol one-step method to extract the total RNA in the transwell lower layer cells of each group;Using beta-actin as a reference,the RT-PCR method was used to detect the expression of nestin and ngn3 m RNA.7 Detect the distribution and expression of Nestin and Ngn3 by immunofluorescence assay After co-culture,the expression of nestin and ngn3 protein in the transwell lower layer cells of each group was detected by immune fluorescence.Results: 1 Morphological changes of primary bone marrow mesenchymal stem cells The freshly isolated cells are spherical shape and suspend in the medium;when change the total liquid after 24 h,the cells begin to adhere to the wall,but most of the cells were still round;some of the cells begin to bulge and stretch out pseudopodia,and showed polygonal and colony growth after 72 h;To the P3,most are fusiform,fibroblast,and whirlpool like growth.2 Morphological changes of primary pancreatic stem cells The freshly isolated cells are suspended in the medium,and because the cells were digested by collagenase V,a large number of fibroblasts were contained.Fibroblasts are easier to attach to the pancreatic stem cells,so they are differential adherenced to 1 h every time;after the differential adherenced 2~3 times,the cells of the first few bottles were adherent to a large number of spindle cells,which were typical fibroblast cells.After that,the cells showed cobblestone like and adherent growth after 24 h.3 Identification of surface markers of bone marrow mesenchymal stem cells by flow cytometry Flow cytometry results showed the expression of CD44,CD90 and CD105 of P3 generation cells were 89.4%,94.5% and 99.8%,while CD34 and CD45 were 9.48%,8.22%.The results showed that the primary cultured cells were bone marrow mesenchymal stem cells.4 Identification of pancreatic stem cells by immunofluorescence The results of immunofluorescence assay showed that the expression of Nestin and Ngn3 protein was significantly increased in P2 pancreatic stem cells which declaring that the primary cultured cells were pancreatic stem cells.5 The expression of nestin and ngn3 m RNA after co-culture Compared with BMSC group,the levels of nestin and ngn3 m RNA in PSCS group and CC group were significantly higher.Compared with PSCS group,the level of nestin m RNA in CC group was significantly lower.These results indicated that the bone marrow mesenchymal stem cells did not express nestin and ngn3 m RNA,but co-cultured cells had the tendency to differentiate into pancreatic stem-like cells.6 The distribution of Nestin and Ngn3 in co-cultured cells Compared with BMSC group,the levels of Nestin and Ngn3 protein were significantly higher in PSCS group and CC group.There was no significant difference between PSCS group and CC group.These results indicated that the induced bone marrow mesenchymal stem cells expressed Nestin and Ngn3 protein,and had the tendency to differentiate into pancreatic stem-like cells.Part two The repair effect of induced bone marrow mesenchymal stem cells on the islet function of diabetes mellitusObjective: Diabetes(Diabetes mellitus DM)is a global epidemic and chronic disease,due to absolute or relative deficiency of insulin or reduction of target cell sensitivity to insulin or insulin itself has a structural defects cause the sugar,fat and protein metabolism disorder,and characterized by elevated blood glucose levels.At present,the treatment of diabetes is mainly hypoglycemic drugs,but the drug treatment did not achieve satisfactory therapeutic effect,just control the blood glucose level,and did not solve the problem of insufficient pancreatic islet function fundamentally.In recent years,with the development of regenerative medicine,stem cell therapy is gradually emerging,which provides a new way to solve the above problems.Bone marrow mesenchymal stem cells(BMSC)are ideal seed cells for tissue engineering,regenerative medicine,and autoimmune diseases.However,the direct transplantation of BMSC often leads to poor therapeutic effect due to lack of targeting.Pancreatic stem cells(PSCS)is a kind of adult stem cells existing in fetal and adult pancreas,which maintaining self-renewal and multiple differentiation potentials.But because of the lack of pancreatic stem cell and other problems such as immune rejection,direct transplantion and transplantation after inducing to islet like cell mass can not achieve the desired therapeutic effect.In this study,we used pancreatic stem cells(PSCS)to induce bone marrow mesenchymal stem cells(BMSC)to differentiate into diabetic rats and then injected by tail vein to explore the repair effect on the islet function of diabetes mellitus.The DM model was reproduced by STZ(streptozotocin)in healthy male SD rats,then every treatment group received intravenous injection of cells.Detecting the blood glucose,body weight changes and glucose tolerance curve,using DIR-iodide staining cells and trace,and rat pancreas were observed under light microscope.Using ELISA kit to detect the serum insulin,C peptide content,using glycogen assay kit to detect rat liver glycogen and muscle glycogen content,using the Western blot method to detect the Insulin protein in rat pancreatic tissue expression.It aims to further explore the overall efficacy and effect of cell reversal of DM after induction,and provide experimental basis for clinical treatment.Methods: 1 Animal grouping and sampling 60 healthy male SD rats were randomly divided into 6 groups,the normal group(Con)of 5,the remaining 55 were injected by STZ solution to prepare DM model.After 3 days of injection,the fasting blood glucose was higher than 16.7mmol/L,so as to make the model successful.The successful model is 45,and then randomly divided into 4 groups: Pathological group(DM,n=9),PBS group(PBS,n = 5),group BMSC(BMSC,n = 15),co-culture group(CC,n = 16).The above 4 groups were fed at room temperature of 20-25.The BMSC group and the co-culture group were injected with 5 × 106 cells in each group,and were injected for 1 times every two weeks.After 5 weeks of feeding,all animals were drugged and executed.2 The blood glucose and body weight Blood samples were taken from the ear vein,and blood glucose levels were measured and recorded before and after injected STZ 1 weeks,2 weeks,3 weeks,4 weeks,and 5 weeks.The changes of body weight were recorded before and after injected STZ 1 weeks,2 weeks,3 weeks,4 weeks,and 5 weeks.3 Changes of glucose tolerance After second injections of cells on the morning of the seventh day,the rats were fasted for 6 hours.After 6 hours,collected the blood in ear vein and detected blood glucose.Then intraperitoneal injected the glucose(100 mg/ml,injection dose given by 1 mg/g weight).Blood glucose was recorded at the time of injection,0.25 h,0.5 h,1 h and 2 h,respectively,and used to describe the glucose tolerance curve.4 Cell labeling and tracing Using DIR-iodide stained cells and photographed the vivo image after the intravenous injection of 7 days.5 The morphological changes of pancreatic tissue were observed under light microscope Pancreatic tissue was fixed with 4% paraformaldehyde,paraffin embedded,sectioned and stained with HE.The morphological changes of pancreas were observed under light microscope.6 Detection of serum insulin in rats by insulin ELISA Kit 7 Detection of C peptide in rat serum by C peptide ELISA Kit 8 Detection of the content of liver glycogen and muscle glycogen by glycogen test kit 9 Detection of the expression of insulin in pancreatic tissue of rats by Western blotResults: 1 The general performance of rats and the changes of blood glucose and body weight The symptoms of diabetic rats include loss of body weight,dull,coarse hair,lethargy,polyuria,polydipsia,polyphagia and growth retardation and so on.With the experiment,the symptoms of diabetic rats were more and more apparent in DM and PBS groups,while the symptoms of diabetic rats had significantly improved in BMSC and CC groups.There was no significant difference in blood glucose and body weight in each group before grouping.Throughout the experiment,there was no overt significance at the average blood glucose level between BMSC and CC(P>0.05),but the two groups were significantly lower than Con group(P<0.01),and significantly higher than those in group DM and group PBS(P<0.01).There was no significant difference between the BMSC group and the CC group(P<0.05),the above two groups were significantly higher than those in group Con,and significantly lower than those in group PBS and group DM(P<0.01).2 Rat glucose tolerance test Compared with Con,DM group,PBS group,BMSC group and CC group significantly increased in the area under the curve of glucose tolerance(P<0.01),and compared with the DM group,PBS group,BMSC group and CC group significantly reduced in the area under the curve of glucose tolerance(P<0.05).3 Cell labeling and tracing In vivo imaging showed that the transplanted cells could be concentrated in the abdomen.4 The morphological changes of pancreas were observed under light microscope Con group: the number of islets was more,the contours were regular,the size of islets was large,the cells arranged in the islets were orderly,the nucleus was central,the cytoplasm was uniform,and the cell boundaries were clear DM and PBS group: the number of islets was reduced to moderate to severe,and the size of pancreatic islets was reduced to moderate to severe,the cells were vacuolar degeneration,and the nucleus was biased to one side,showed apoptosis.CC group: the number of islets was more,the islet volume was decreased slightly,the number of islet cells was decreased slightly,and the vacuolar degeneration was observed,the cytoplasm was even.5 ELSIA detection of serum insulin and C peptide levels in rats Compared with Con group,the levels of insulin and C peptide in the DM group,PBS group,BMSC group and CC group were significantly decreased,and there was statistical difference.Compared with BMSC group,the insulin level in CC group was significantly higher.6 Detection of the content of liver glycogen and muscle glycogen by glycogen test kit Compared with Con group,the liver glycogen and muscle glycogen levels in DM group,PBS group,BMSC group,CC group were significantly reduced,there were statistically significant differences.Compared with BMSC group,the level of liver glycogen was significantly higher in CC group,but there was no significant difference between the BMSC group and the control group.7 Detection of the expression of insulin in pancreatic tissue of rats by Western blot Compared with Con group,the insulin level was significantly lower than that in DM group and PBS group,.Compared with Con group,insulin level was not significantly different from CC group.Conclusions: 1 Co-cultured with pancreas stem cells using transwell,bone marrow mesenchymal stem cells were induced to differentiate into pancreatic stem-like cells.2 BMSC can effectively improve the condition of diabetic rats.3 The induced bone marrow mesenchymal stem cells have a better targeting effect on the repair of diabetic islet function.