| Objective:(1) To observe the effect of Glucagon like peptide-1 receptor agonist liraglutide combined human umbilical cord mesenchymal stem cells on the glucose metabolism and the islet βcell function of type 2 diabetes mellitus SD rats.And then to investigate relation of the effect on the glucose metabolism and the islet βcell function between the inflammatory signal pathway mechanism.(2) Design prospective small sample clinical study:A 24 week, randomized, single blindness comparison to value the effect ofcombined human umbilical cord mesenchymal stem cells on the glucose metabolism and the islet βfunction of type 2 diabetes mellitus patients whowere poor efficacy by Glucagon like peptide-1 receptor agonist liraglutide.Method:(1) Type 2 diabetes (T2DM) rats were established by giving high-carbonhydrate-fat diet 4 weeks andthen injecting 30mg/kg streptozotocin into intraperitoneal.The standard of eligible T2DM model rats was by measuring two times of fasting blood glucose no less than 11.1mmol/L. Then eligible T2DM model rats were randomly divided into four groups according the table of random number:control group (DM), hUC-MSCs treatment group (hUC-MSCs), Liraglutide treatment group (LIRA), Liraglutide combined with hUC-MSCs treatment group(LIRA+hUC-MSCs). The number of rats in each group is ten.Then give the appropriate drug therapy 8 weeks respectively. Rats of DM group were given 100ul cell nutrient solution without human umbilical cord mesenchymal stem cells by caudal vein injection and 2ml normal saline by subcutaneous injection twice a day.Rats of hUC-MSCs group were given 100ul cell nutrient solution with human umbilical cord mesenchymal stem cells by caudal vein injection and 2ml normal saline by subcutaneous injection twice a day. T2DM Rats of LIRA group were given 100ul cell nutrient solution without human umbilical cord mesenchymal stem cells by caudal vein injection and 2ml liraglutide diluent200ug/kgby subcutaneous injection twice a day.Rats of LIRA+hUC-MSCs group were given 100ul cell nutrient solution with human umbilical cord mesenchymal stem cells by caudal vein injection and 2ml liraglutide diluent200ug/kgby subcutaneous injection twice a day.During the treatment period, the general condition indexs of each group rats were monitored. The serum FBG, HbAlc, C-P, GLu, GLP-1,GAS and CCK were measured respectively. The changes of pancreatic tissue pathology were observed through HE stain under microscope.The expressions of insulin and glucagon in pancreas tissue were surveyingthrough immunohistochemistry coloration.The apoptosis status of the Islet cells in pancreas tissue wereevaluatedthrough TUNEL. And the expressions of the NF-κB mRNA and TLR4 mRNAin pancreas tissue were determined by Real time polymerase Chain Reaction.(2) Adopting randomized, single blindness, comparisonprospective small sample clinical study. Screening of eligible subjects:Type 2 diabetes mellitus patients who were diagnosed according to the rule of WHO(1999) and having a more than 10 years duration, withpoor efficacy (7%≤HbA1c≤10%) by Glucagon like peptide-1 receptor agonist liraglutideunder using secretagogue, metformin and premixed or basal insulin analogues, the daily dose of secretagogue must no less than half of recommended dose,the daily dose of metformin must no less than 1000mg, the daily dose of insulin must no less than 20U, liraglutide was given by subcutaneous injection 1.2mg at bedtime per day. The treatment must maintain dimensional for less than 6 months.Every eligible subjects must sign the clinical study of informed consent. Twelve patients were randomly divided into two groups:the group of liraglutide combined false transplanting human umbilical cord mesenchymal stem cells treatment (LIRA-hUC-MSCsgroup) and the group of liraglutidecombined transplanting human umbilical cord mesenchymal stem cells treatment (LIRA+hUC-MSCs group).Each group has 6 patients. Liraglutide was sequentially given by subcutaneous injection 1.2mg at bedtime per day for 24 weeks.The programs of transplanting human umbilical cord mesenchymal stem cells treatment was followed by infusing 1×106/kg cells through pancreatic artery directed the interventional radiology on the first day, and by infusing 1×106/kg cells through peripheral vein on the eighth day, the fifteenth day and the twenty-second day sequentially. The programs of false transplanting human umbilical cord mesenchymal stem cells treatment was followed by infusing the same capacity normal saline through pancreatic artery directed the interventional radiology on the first day, and by infusing normal saline through peripheral vein on the eighth day, the fifteenth day and the twenty-second day sequentially. During the treatment period, the general condition indexs of each group patients were monitored. The FBGand HbA1 cwere measured. Carrying on 75g oral glucose tolerance test to estimate the early phase of C peptide secretion function (△Cp30/△G30), the total amount of C peptide secretion function (area under the curve of C peptide within 180 minutes, AUCCP180) and Homeostasis model assessment of insulin resistance (HOMA-IR=FIns×FPG/22.5).Results:(1) Before randomized treatment, the FBG and HbA1c were comparable between four groups. After 8weeks treatment, compared with DM group, the FBG and HbA1c of three treatment groups were significantly decreased (P<0.05, P< 0.01). On the mean time, compared with hUC-MSCs group, the HbA1c ofLIRA group and LIRA+hUC-MSCs group were significantly decreased (P<0.01). Compared sequentially with LIRA group, the HbA1c of LIRA+hUC-MSCs group were also significantly decreased (P<0.05)(2) After 8weeks treatment, the C-P, GLu, GLP-1, GAS and CCK were measured.Compared with DM group, the C-PofhUC-MSCs group was significantly increased;the C-P, GLP-1,GAS and CCK of LIRA group and LIRA+hUC-MSCs group were significantly increased while the GLu were significantly decreased (P <0.05;. Compared with hUC-MSCs, the C-P, GLP-1 of LIRA group and LIRA+hUC-MSCs group were significantly increased while the GLu were significantly decreased (P<0.05). Compared sequentially with LIRA group, the theC-P, GLP-1 of LIRA+hUC-MSCs group were also significantly increased while the GLu were significantly decreased (P<0.05)(3) After 8weeks treatment, the changes of pancreatic pathology were observed by HE stain under microscope. In the DM group,islet disorder with little cell unite could be observed. Compared with the DM group, islet architecture and cell unite amount were significantly ameliorated in the three treatment groups. Bu the diffenence of morphology among the three treatment groups could not be observed. The expressions of insulin and glucagon in pancreas tissue were surveying through immunohistochemistry coloration.Compared with hUC-MSCs group or LIRA group, the ratio of insulin positive area in pancreas tissue was obviouslyrised, while the ratio of glucagon positive area in pancreas tissue wasclearlydescend in LIRA+hUC-MSCs group (P<0.05). And the same difference in valuating islet cells apoptosis by TUNEL could be observed. The expression of NF-κB mRNA and TLR4 mRNA in pancreas tissue of LIRA+hUC-MSCs group were the least among the four groups.(4) 12 eligible type 2 diabetes mellitus patients were Twelve patients were randomly divided into two groups:the group of liraglutide combined false transplanting human umbilical cord mesenchymal stem cells treatment (LIRA-hUC-MSCs group) and the group of liraglutide combined transplanting human umbilical cord mesenchymal stem cells treatment(LIRA+hUC-MSCs group). All patients were completed the clinical study.(5) On the baseline, characteristicsincluding body weight, body mass index, weist, blood systolic pressure and diastolic pressure of patients were comparable in the two groups (P>0.05). Compared with the baseline, characteristics of patientsafter24 weeks treatment were also comparable in the two groups (P> 0.05)(6) On the baseline, the FBG,2hPBG and HbA1 cof patients were comparable in the two groups (P>0.05). Compared with the baseline, the FBG,2hPBG and HbA1c were no difference after24 weeks treatment in LIRA-hUC-MSCs group, while the FBG,2hPBG and HbA1c were significantly decreased after24 weeks treatment in LIRA+hUC-MSCs group (P<0.05). Compared with LIRA-hUC-MSCs group, the FBG,2hPBG and HbA1c were significantly decreased after24 weeks treatment in LIRA+hUC-MSCs group (P<0.05)(7) On the baseline, theACp30/AG30, AUCCP 180 and HOMA-IR of patients were comparable in the two groups (P>0.05).Compared with the baseline, the △Cp30/△G30, AUCCP180 and HOMA-IR were no difference after24 weeks treatment in LIRA-hUC-MSCs group, while the △Cp30/△G30, AUCCP180were significantly increased and HOMA-IR was significantly decreased after24 weeks treatment in LIRA+hUC-MSCs group (P<0.05). Compared with LIRA-hUC-MSCs group, the △Cp30/△G30, AUCcp180were significantly increased and HOMA-IR was significantly decreased after 24 weeks treatment in LIRA+hUC-MSCs group (P< 0.05)Conclusion:(1) Compared with a single treatment of liraglutide or transplanting human umbilical cord mesenchymal stem cells, the effect of improving glucose metabolism and the islet βcell function on type 2 diabetes mellitus SD rats were so much the better in the combined treatment of liraglutide and transplanting human umbilical cord mesenchymal stem cells. The indexs of islet βcell function conclude the islet architecture, the expressions of insulin and glucagon in pancreas tissue through immunohistochemistry coloration,and the situation of islet cells apoptosis by TUNEL. And it also shows that effect of ameliorating on glucose metabolism and the islet βcell function with giving combined treatment to type 2 diabetes mellitus SD rats was closely related with down regulating the TLR4/NF-κB inflammatory signling pathway.(2) The 24 week, randomized, single blindness prospective small sample clinical study shows that combining human umbilical cord mesenchymal stem cells, the effect of improving glucose metabolism and the islet βcell function was so much the better on type 2 diabetes mellitus patients who were poor efficacy by glucagon like peptide-1 receptor agonist liraglutide. |
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