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Objective • To investigate changes of immune thrombocytopenia (ITP) patients-derived bone marrow mesenchymal cells (BMCs) in cells survival, cytokines expression as well as the effects of BMCs on the biological behaviors of megakaryocytes. Methods • BMCs were collected from 7 ITP patients and 5 normal controls (NC), and cultivated by the whole marrow adherent method. Surface markers and basal apoptosis rate of BMCs were analyzed by flow cytometry (FCM). Proliferation of BMCs was assessed by CCK-8 method. Phorbol 12-myristate 13-acetate (PMA) was used to stimulate differentiation of HEL cells. The induced HEL cells (inHEL) were divided into 3 groups: inHEL cultured alone (group a), inHEL co-cultured with BMCs derived from ITP patients (group b), inHEL co-cultured with BMCs derived from NC (group c). After 72 h incubation, the expression of cell surface proteins (CD41a, CD42b) and cell apoptosis rate were analyzed by FCM. The mRNA and proteins expression levels of cytokines IL6, IL11, TPO, SCF were detected by real-time fluorescent quantitative PCR (RT-qPCR) and enzyme linked immunosorbent assay (ELISA), respectively. Results • Compared with NC, BMCs from ITP patients grew progressively slowly (Day 4, P=0.039; Day 6, 10, P=0.009; Day 8, P=0.007), cell basal apoptosis rates were increased [AV+PI- (early apoptosis rate), P=0.036; AV+PI+ (late apoptosis rate), P=0.003; AV+PI-/+ (total apoptosis rate), P=0.004]. Compared with group a, the expression of CD41a in group c was much higher (P=0.000). The expression of CD41a in group b was higher than that in group a (P=0.015), but still much less than that in group c (P=0.000). Compared with group a, the early and total apoptosis rate in group b, c and the late apoptosis rate in group c were decreased obviously (all P=0.000), whereas there was no obvious change of the late apoptosis rate in group b. However, compared with group c, the late and total apoptosis rate in group b were significantly increased (both P=0.000). The expression levels of IL6, SCF mRNA and IL6 protein were significantly decreased in ITP BMCs (all P=0.000), but there was no obvious difference in the expression levels of IL11 and TPO between ITP BMCs and NC BMCs. Conclusion • BMCs from ITP patients show some defects in supporting megakaryocytic differentiation and survival under co-culture conditions, which mechanisms are related to the reduction of IL6 and SCF expression.
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Objective·To investigate changes of immune thrombocytopenia (ITP) patients-derived bone marrow mesenchymal cells (BMCs) in cells survival, cytokines expression as well as the effects of BMCs on the biological behaviors of megakaryocytes. Methods?·?BMCs were collected from 7 ITP patients and 5 normal controls (NC), and cultivated by the whole marrow adherent method. Surface markers and basal apoptosis rate of BMCs were analyzed by flow cytometry (FCM). Proliferation of BMCs was assessed by CCK-8 method. Phorbol 12-myristate 13-acetate (PMA) was used to stimulate differentiation of HEL cells. The induced HEL cells (inHEL) were divided into 3 groups: inHEL cultured alone (group a), inHEL co-cultured with BMCs derived from ITP patients (group b), inHEL co-cultured with BMCs derived from NC (group c). After 72 h incubation, the expression of cell surface proteins (CD41a, CD42b) and cell apoptosis rate were analyzed by FCM. The mRNA and proteins expression levels of cytokines IL6, IL11, TPO, SCF were detected by real-time fluorescent quantitative PCR (RT-qPCR) and enzyme linked immunosorbent assay (ELISA), respectively. Results?·?Compared with NC, BMCs from ITP patients grew progressively slowly (Day 4, P=0.039; Day 6, 10, P=0.009; Day 8, P=0.007), cell basal apoptosis rates were increased [AV+PI- (early apoptosis rate), P=0.036; AV+PI+(late apoptosis rate), P=0.003; AV+PI-/+(total apoptosis rate), P=0.004]. Compared with group a, the expression of CD41a in group c was much higher (P=0.000). The expression of CD41a in group b was higher than that in group a (P=0.015), but still much less than that in group c (P=0.000). Compared with group a, the early and total apoptosis rate in group b, c and the late apoptosis rate in group c were decreased obviously (all P=0.000), whereas there was no obvious change of the late apoptosis rate in group b. However, compared with group c, the late and total apoptosis rate in group b were significantly increased (both P=0.000). The expression levels of IL6, SCF mRNA and IL6 protein were significantly decreased in ITP BMCs (all P=0.000), but there was no obvious difference in the expression levels of IL11 and TPO between ITP BMCs and NC BMCs. Conclusion?·?BMCs from ITP patients show some defects in supporting megakaryocytic differentiation and survival under co-culture conditions, which mechanisms are related to the reduction of IL6 and SCF expression.
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Objective To explore the effect and the mechanism of vitamin D(Vit D) promotes proliferation and differentiation of mesenchymal stem cells (MSCs) through regulates extracts of plastrum testudinis (PTE).Methods Established the PGL3-Id1 promoter and transfered rat MSCs.PTE combined with 10-6,10-7,10-8mol/L Vit D respectively were acted on the transfected MSCs for 36 hours.The level of Id1 promoter were detected by luciferase activity measurement.1,3,30,100 pg/mL PTE combined with Vit D of 10-7 mol/L were acted on MSCs for 36 hours,3 days and 7 days,and the VDR expression were detected by RT-PCR test.Results PTE promoted the expression of Id1 in MSCs,the expression of Id1 was inhibited when PTE combined with Vit D (P ＜ 0.01),and it was significantly different among different dosis of Vit D(P ＜0.01).The expression of VDR was inhibited in different degree when PTE combined with Vit D for 36 hours,3 days and 7 days.PTE combed with large dose of Vit D for 36 hours had significant effect of inhibition,and the difference was statistically significant (P ＜ 0.05).The inhibiting effect was more obvious when PTE combined with large dose of Vit D for 3 days and 7 days.When different doses of PTE combined with Vit D for a same duration,the difference of VDR expression was statistically significant (P ＜ 0.05).Meanwhile,when same doses of PTE combined with Vit D for different durations,the difference of VDR expression at 7 days and 36 hours was statistically significant (P ＜ 0.05).Conclusion The proliferation of MSCs which promoted by PTE was inhibited by Vit D,and the nuclear receptor VDR may be one of the targets of drug action for PTE regulating proliferation and differentiation of MSCs.
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Objective To investigate whether pancreatic islet transplantation in combination with bone mesenchymal stem cells (MSC) transplantation can promote the vascularization surrounding the transplant pancreatic islet.Methods The non-obese diabetic (NOD) mice were utilized as the recipients and randomly divided into pancreatic islet transplantation combined with MSC transplantation group (co-transplantation group,n=6),pancreatic islet transplantation group(n=6),MSC transplantation group(n=6) and sham transplantation group (n=3).The variation in blood glucose level and survival rate post-transplantation of NOD mice in each group was observed.The proliferation and apoptosis of the transplant pancreatic islet in the pancreatic islet transplantation group and co-transplantation group at 1,2 and 4 weeks after pancreatic islet transplantation were analyzed by 5-ethynyl-2'-deoxyufidine (EdU) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL).The vascular density surrounding the transplanted pancreatic islet in the pancreatic islet transplantation group and co-transplantation group at postoperative 2,4 and 8 weeks were observed under light microscope and quantitatively analyzed by histochemical and immunohistochemical staining.Results Both MSC combined with pancreatic islet transplantation and pancreatic islet transplantation significantly improved the blood glucose level and enhanced the survival rate of NOD mouse models after transplantation.In addition,it could accelerate the regeneration of pancreatic islet cells and decrease cell apoptosis.MSC combined with pancreatic islet transplantation significantly enhanced the vascular density surrounding the transplant pancreatic islet compared with pancreatic islet transplantation alone.Conclusions MSC transplantation can accelerate the vascularization surrounding the transplant pancreatic islet,increase the blood supply and protect the function and activity of the transplant pancreatic islet.
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Bone marrow contains mesenchymal stem cells capable of differentiating into osteoblastic lineage. To test the ability of purified bone marrow mesenchymal cell loaded onto xenograft with type I collagen gel to heal bone defect, mesenchymal stem cells were isolated from bone marrow of Lewis rat, and culture-expanded. Northern blot hybridization revealed that osteocalcin and osteopontin mRNA expression persisted for 8 weeks of in-vitro culture. When the bone marrow mesenchymal cells became 80-90% confluent in 2-3 weeks, they were loaded onto chips of xenograft (Lubboc(R) with type I collagen gel. The cell-gel-xenograft composite was transplanted subcutaneously into the dorsum of nude mice. It was also transplanted into tibial diaphyseal defect made in syngeneic Lewis rats, and compared with the control group where only the xenograft was planted. There was no ectopic bone formation at the dorsum of nude mice around the transplanted cell-gel-xenograft complex. In the long bone defect model, the cell-gel-xenograft group induced fibrosis around it and the bone healing was inferior to the control group. Type I collagen gel is not a suitable carrier of bone marrow mesenchymal cells to heal the long bone defect.