ObjectiveTo determine the expression level of Sonic hedgehog (Shh) in the passage of hair follicle stem cells (HFSCs), analyze the effect of Shh overexpression on the proliferation activity of HFSCs, and explore the survival of HFSCs after Shh overexpression and its effect on hair follicle regeneration. MethodsHair follicles from the normal area (H1 group) and alopecia area (H2 group) of the scalp donated by 20 female alopecia patients aged 40-50 years old were taken, and the middle part of the hair follicle was cut under the microscope to culture, and the primary HFSCs were obtained and passaged; the positive markers (CD29, CD71) and negative marker (CD34) on the surface of the fourth generation HFSCs were identified by flow cytometry. The two groups of HFSCs were transfected with Shh-overexpressed lentivirus. Flow cytometry and cell counting kit 8 assay were used to detect the cell cycle changes and cell proliferation of HFSCs before and after transfection, respectively. Then the HFSCs transfected with Shh lentivirus were transplanted subcutaneously into the back of nude mice as the experimental group, and the same amount of saline was injected as the control group. At 5 weeks after cell transplantation, the expression of Shh protein in the back skin tissue of nude mice was detected by Western blot. HE staining and immunofluorescence staining were used to compare the number of hair follicles and the survival of HFSCs between groups. ResultsThe isolated and cultured cells were fusiform and firmly attached to the wall; flow cytometry showed that CD29 and CD71 were highly expressed on the surface of the cells, while CD34 was lowly expressed, suggesting that the cultured cells were HFSCs. The results of real-time fluorescence quantitative PCR and Western blot showed that the expression levels of Shh protein and gene in the 4th, 7th, and 10th passages of cells in H1 and H2 groups decreased gradually with the prolongation of culture time in vitro. After overexpression of Shh, the proliferation activity of HFSCs in the two groups was significantly higher than that in the blank group (not transfected with lentivirus) and the negative control group (transfected with negative control lentivirus), and the proliferation activity of HFSCs in H1 group was significantly higher than that in H2 group before and after transfection, showing significant differences (P<0.05). At 5 weeks after cell transplantation, Shh protein was stably expressed in the dorsal skin of each experimental group; the number of hair follicles and the expression levels of HFSCs markers (CD71, cytokeratin 15) in each experimental group were significantly higher than those in the control group, and the number of hair follicles and the expression levels of HFSCs markers in H1 group were significantly higher than those in H2 group, and the differences were significant (P<0.05). ConclusionLentivirus-mediated Shh can be successfully transfected into HFSCs, the proliferation activity of HFSCs significantly increase after overexpression of Shh, which can secrete and express Shh continuously and stably, and promote hair follicle regeneration by combining the advantages of stem cells and Shh.
Objective To observe the effects of the bone marrow mesenchymal stem cells (BMSCs) on the expression of neurotrophic factor protein gene in the retinal detachment (RD) rabbits. Methods 60 healthy rabbits were randomly divided into control group (group A), retinal detachment with PBS group (group B), retinal detachment with BMSCs group (group C), 20 rabbits in each group. RD model were established for rabbits in group B and C. 10 μl PBS was injected into the subretinal space of rabbits in group B, while 10 μl CM-Dil labeled BMSC PBS was injected into subretinal space of rabbits in group C. The rabbits in the group A received no treatment. At 1, 2 and 4 weeks after modeling, the mRNA expression of basic fibroblast growth factor (bFGF), brain derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) were measured by real-time quantitative PCR. Results At 1, 2 and 4 weeks after modeling, the mRNA expression of bFGF, BDNF, CNTF on retinal tissue were increased significantly in group C as compared with group A and B (P < 0.01). At 1 week after modeling, the mRNA expression of bFGF and CNTF on retinal tissue were increased significantly in group B as compared with group A, the mRNA expression of BDNF on retinal tissue in group B was similar with group C. At 2 and 4 weeks after modeling, the mRNA expression of bFGF, BDNF, CNTF were decreased in group B as compared with group A. Conclusion Subretinal transplantation of BMSC can increase the mRNA expression of bFGF, BDNF and CNTF on retinal tissue in RD rabbits.
Objective To observe the survival of human umbilical cord derived mesenchymal stem cells (hUC-MSCs) after injection into the vitreous of rabbits,and the animal safety under those procedures.Methods Twentyseven pigmented rabbits were randomly divided into 3 groups (intravitreal injection 1 week group，2 weeks group and 4 weeks group)， each with 9 rabbits.For each animal the right eye was the experimental eye receiving hUCMSCs injection,while the left eye was the control eye receiving culture medium. The rabbit eyes were examined by slitlamp microscope, indirect ophthalmoscopy, fundus photography, fundus fluorescence angiography（FFA）and Tonopen tonometer before and after injection. hUCMSCs were labeled by CMDil in vitro, and their survival status was measured by confocal fluorescence microscopy, light microscope and transmission electron microscope at 4 weeks after injection. Results Four weeks after injection, a large number of the hUCMSCs were still alive in the vitreous cavity. The overall condition of those rabbits was good. The anterior segment and retina of experimental eyes were normal, without hyperfluorescence, hypofluorescence and leakage in the retina at 1,2 and 4 weeks after injection. There was no significant difference on IOP before and after injection at different time points (P＞0.05), and no obvious changes at cornea, anterior chamber angle,lens,retinal structure by.light microscope and transmission electron microscope examination.Conclusion hUC-MSCs can survive in the rabbit vitreous for four weeks；intravitreal injection of hUCMSCs was safe and feasible.
Objective To evaluate the effect of smooth muscle cell transplantation on myocardial interstitial reconstruction shortly after myocardial infarction. Methods A total of 48 female Wister rats were randomly divided into two groups with the random number table, the control group (n=24) and the smooth muscle cell transplantation group (n=24). The left coronary artery was ligated to set up the myocardial infarction animal model. An amount of 05 ml phosphate buffered saline(PBS) containing 1×106 smooth muscle cells or 0.5 ml PBS without cells was injected into the injured myocardium immediately. By immunoblot and reverse transcriptionolymerase china reaction (RT-PCR), we observed the amount of protein and mRNA of matrix metalloproteinase2(MMP-2), matrix metalloproteinase-9(MMP-9) and tissue inhibitor of metalloprotease-3 (TIMP-3) in the myocardium of the rats. Results The transplanted smooth muscle cells survived well. Compared with the control group, myocardial TIMP3 mRNA (1.06±0.22 vs. 0.81±0.19, t=－2.358, P=0.033) and protein content (3.33±0.53 vs. 1.63±0.47, t=－6.802, Plt;0.001) were significantly increased in the transplantation group. Myocardial MMP-2, MMP-9 mRNA (0.49±0.12 vs. 1.16±0.18, t=8.453, Plt;0.001; 0.45±0.12 vs. 0.80±0.11, t=5.884, Plt;0.001) and protein content (3.98±1.08 vs. 6.05±0.91, t=4.139, P=0.001; 0.39±0.14 vs. 0.57±0.17, t=2.409, P=0.031) [CM(1585mm]were significantly reduced in the transplantation group compared with the control group. Conclusion transplanted smooth muscle cells can survive well in the infarction myocardium and can increase the amount of myocardial TIMP-3 mRNA and protein content and reduce myocardial MMP-2, MMP-9 mRNA and protein content, which is an effective way to prevent harmful cardiac remodeling.
ObjectiveTo analyze the efficacy and safety of various treatment strategies for patients with refractory/recurrent diffuse large B-cell lymphoma (r/r-DLBCL) by network meta-analysis. MethodsThe PubMed, EMbase and Cochrane Library databases were searched to collect randomized controlled trials (RCTs) and clinical controlled trials related to the objectives of the study from inception to November 16th, 2022. After two investigators independently screened the literature, extracted data and evaluated the risk of bias of the included studies, a network meta-analysis was performed using R 4.2.2 software. ResultsA total of 8 RCTs and 11 non-randomized controlled trials were included, involving 2 559 cases. The treatment regimen included chemotherapy, immunochemotherapy, chemotherapy combined with ADC, immunochemotherapy combined with ADC, ASCT based regimen, CAR-T based regimen, ASCT combined with CAR-T, immunomodulators, small molecule inhibitors, and rituximab combined with small molecule inhibitors. The ranking probability results showed that the top three complete remission (CR) rates among all schemes were ASCT combined with CAR-T, chemotherapy combined with ADC, and immune modulators; The top three overall response rates (ORR) were chemotherapy combined with ADC, ASCT combined with CAR-T, and ASCT. The CAR-T regimen had a higher rate of severe neutropenia; The severe thrombocytopenia rate of ASCT regimen was relatively high; There was no significant difference in the incidence of SAEs among the other options. ConclusionASCT combined with CAR-T and chemotherapy combined with ADC have the best therapeutic effects on r/r-DLBCL. However, the specific protocol to be adopted requires clinical doctors to combine actual conditions, comprehensively consider the efficacy and side effects, and develop personalized treatment strategies for r/r-DLBCL patients.
Stargardt disease (STGD) is one of the most prevalent inherited macular dystrophy, and most often occurs in child or adolescence. Irreversible vision loss is observed in almost all cases. Type 1 (STGD1) is one of the most common type. It is an autosomal recessive condition, caused by mutations in the Abca4 gene. In recent years, encouraging progress has been made in the treatment of STGD1. C20-D3-retinyl acetate (ALK-001), fenretinide and ICR-14967 (A1120) as visual cycle modulators, StarGen as gene supplementation therapies, and the stem cell transplantation of human embryonic stem cell-derived retinal pigment epithelium cells are the most promising therapies. With the development of studies and clinical trials, the clinical application of various treatments of STGD1 are expected in the near feature, which are expected to save the vision of most patients.
Objective To investigate the effect of M2 microglia (M2-MG) transplantation on spinal cord injury (SCI) repair in mice. Methods Primary MG were obtained from the cerebral cortex of 15 C57BL/6 mice born 2-3 days old by pancreatic enzyme digestion and identified by immunofluorescence staining of Iba1. Then the primary MG were co-cultured with interleukin 4 for 48 hours (experimental group) to induce into M2 phenotype and identified by immunofluorescence staining of Arginase 1 (Arg-1) and Iba1. The normal MG were harvested as control (control group). The dorsal root ganglion (DRG) of 5 C57BL/6 mice born 1 week old were co-cultured with M2-MG for 5 days to observe the axon length, the DRG alone was used as control. Forty-two 6-week-old female C57BL/6 mice were randomly divided into sham group (n=6), SCI group (n=18), and SCI+M2-MG group (n=18). In sham group, only the laminae of T10 level were removed; SCI group and SCI+M2-MG group underwent SCI modeling, and SCI+M2-MG group was simultaneously injected with M2-MG. The survival of mice in each group was observed after operation. At immediate (0), 3, 7, 14, 21, and 28 days after operation, the motor function of mice was evaluated by Basso Mouse Scale (BMS) score, and the gait was evaluated by footprint experiment at 28 days. The spinal cord tissue was taken after operation for immunofluorescence staining, in which glial fibrillary acidic protein (GFAP) staining at 7, 14, and 28 days was used to observe the injured area of the spinal cord, neuronal nuclei antigen staining at 28 days was used to observe the survival of neurons, and GFAP/C3 double staining at 7 and 14 days was used to observe the changes in the number of A1 astrocytes. Results The purity of MG in vitro reached 90%, and the most of the cells were polarized into M2 phenotype identified by Arg-1 immunofluorescence staining. M2-MG promoted the axon growth when co-cultured with DRGs in vitro (P<0.05). All groups of mice survived until the experiment was completed. The hind limb motor function of SCI group and SCI+M2-MG group gradually recovered over time. Among them, the SCI+M2-MG group had significantly higher BMS scores than the SCI group at 21 and 28 days (P<0.05), and the dragging gait significantly improved at 28 days, but it did not reach the level of the sham group. Immunofluorescence staining showed that compared with the SCI group, the SCI+M2-MG group had a smaller injury area at 7, 14, and 28 days, an increase in neuronal survival at 28 days, and a decrease in the number of A1 astrocytes at 7 and 14 days, with significant differences (P<0.05). ConclusionM2-MG transplantation improves the motor function of the hind limbs of SCI mice by promoting neuron survival and axon regeneration. This neuroprotective effect is related to the inhibition of A1 astrocytes polarization.
ObjectiveTo observe the influence of human umbilical cord mesenchymal stem cells (hUCMSC) transplantation into vitreous cavity of diabetic rats on the retinal morphology, and the expression of glial fibrillary acidic protein (GFAP) and rhodopsin (RHO). Methods78 male Sprague-Dawley rats were used. 70 rats were injected with streptozotocin by tail vein injection at a dose of 40 mg/kg to establish the diabetes mellitus model, and another 8 rats were injected with 0.1 mol/L pH 4.0 citric acid buffer at the same dose as the normal control group. After 6 weeks of modeling, 10 rats were taken as the control group of diabetic model. hUCMSC suspension was injected into the right eye vitreous cavity of the remaining 60 rats, and the same volume of Dulbecco's modified Eagle/F12 medium was injected into the left vitreous cavity as control eyes. 1, 2 and 4 weeks after transplantation, follow-up experiments were performed. The experimental eyes were labeled as U1, U2, and U4 groups, while the control eyes were recorded as D1, D2, D4, and each group consisted of 20 eyes. After paraffin section and hematoxylin-eosin staining, the structure of the retina was observed by optical microscopy and the thickness of the outer nuclear layer and the inner nuclear layer (INL) were measured. The distribution and migration of hUCMSC in rat retina were observed by frozen section-tissue immunofluorescence assay. The mRNA and protein expression of GFAP and RHO in the retina were detected by real-time quantitative polymerase chain reaction (PCR) and Western blot assays. ResultsThe results of optical microscope observation showed the normal structure of retina in normal control group. The retinal nerve fiber layer (NFL) was thinned and the number of retinal ganglion cells (RGC) in the control group of diabetic rats was decreased. The decreased number and disorder arrangement of RGC were observed as well in U1, D1 rats. The RGC number of U2, U4, D2, D4 rats was gradually decreased. Compared with D4 group, the thickness of INL in U4 group was significantly increased (P < 0.05). Tissue immunofluorescence assay showed that hUCMSC were distributed along the inner limiting membrane in the retina of the U1 group, while the number of hUCMSC in the U2 group was gradually decreased, mainly in the NFL and ganglion cell layers. Real-time PCR and Western blot data indicated that the relative expression of GFAP mRNA and protein in the diabetic retina was significantly increased, and the relative expression of RHO mRNA and protein decreased gradually in the diabetic model group and the D1, D2, D4 groups. Compared with D2 and D4 groups, the mRNA and protein expression of GFAP in U2 and U4 groups were decreased, and the relative expression of RHO mRNA and protein were all increased (P < 0.01). ConclusionhUCMSC could migrate and integrate into the retina, after the transplantation into the vitreous cavity of diabetic rats, which reduced the expression of GFAP, but enhanced the expression of RHO.
Replacement therapy of stem cells transplantation represents a potential treatment for neural retinal diseases. Despite the encouraging results in laboratory, the clinical application of cells replacement therapy is still difficult because the limitation of seed cells, immunologic rejection, oncogenicity and ethical problems, etc. Recent breakthrough in somatic reprogramming provides a promising solution overcoming these obstacles. Further researches on virus free reprogramming will make the clinical application of stem cell replacement therapy possible.
ObjectiveTo summarize the advances of precl inical research in xenogeneic (porcine) cell transplantation in recent years. MethodsThe literature about the precl inical research in xenogeneic (porcine) cell transplantation was analyzed and summarized. ResultsWith the application of new immunosuppressive agents and the generation of transgenic pigs, great progress has been achieved in xenogeneic transplantation of pig-derived nerve cells, islet cells, liver cells, and various types of stem cells. The survival time of xenogeneic cell (porcine) significantly prolonged, but there is still a long way to go before cl inical application. ConclusionThe source of xenogeneic (porcine) cells is abundant and the experiments are reproducible. However, how to effectively prevent rejection and prolong the survival time in the host, and avoid the spread of virus between species are still need to be solved in the future research.