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OBJECTIVE@#To 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.@*METHODS@#Hair 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.@*RESULTS@#The 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).@*CONCLUSION@#Lentivirus-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.
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Animais , Feminino , Camundongos , Alopecia/cirurgia , Folículo Piloso , Proteínas Hedgehog/genética , Camundongos Nus , Regeneração , Células-TroncoRESUMO
Objective To compare different regimens of intraperitoneal injection of cyclophosphamide (CTX) to establish a rabbit model of premature ovarian failure (POF), and provide a useful experimental tool for further research of premature ovarian failure. Methods A total of twenty-one 5-6 months old rabbits were randomly divided into 4 groups. The group A (normal control group) included 3 rabbits without any treatment. The group B (the first model group) included 6 rabbits, received a single intraperitoneal injection of 50 mg/kg cyclophosphamide. Six rabbits in the group C (the second model group) were injected with 50 mg/kg cyclophosphamide once daily for 2 days. The group D (the third model group, also n=6) was injected with 50 mg/kg cyclophosphamide on the first day and then followed by 8 mg/ (kg·d) injection q.d. in the 14 consecutive days. Body weight and ovary weight of the rabbits in each group were measured, and the changes of body weight and the ovary index were analyzed. Morphological changes of the ovarian follicles were observed by HE staining and the numbers of normal and abnormal follicles at different developmental stages were counted and analyzed. Cell apoptosis was analyzed by TUNEL staining and changes in the serum levels of estradiol (E2) were detected by ELISA. Results The body weight of rabbits in both groups B and group C was not significantly changed during the experimental period (P＞ 0. 05). Rabbits in the group D showed a slight growth (P ＜ 0. 05) and high mortality. The ovary index in group C was significantly lower than that in the group A (P ＜ 0. 05). The ratios of abnormal primordial and primary follicles in groups B and C were significantly increased (P ＜ 0. 017), and the ratio of abnormal primordial follicles in the group C was increased more significantly (P ＜ 0. 017). However, there was no significant difference in the ratios of abnormal primary follicles between groups B and C (P＞ 0. 017). Among the groups A, B and C, there was no significant difference in the ratios of abnormal secondary and antral follicles (P＞ 0. 05). Apoptosis mainly occurred in granulosa cells of the ovarian follicles. The apoptosis rate of groups B and C was significantly higher than that in the group A (P ＜ 0. 05), and the apoptosis rate of group C was higher than that in the group B (P ＜ 0. 05). In the group B, the serum E2 level reached the peak value on the 7th day, significantly higher than that on the 35th day (P ＜ 0. 05), and then the level was decreased gradually. In the group C, the E2 level was continuously decreased and the level on the last day before drug injection was significantly higher than that at the 35th day (P ＜ 0. 05). Conclusions Intraperitoneal injection of 50 mg/kg cyclophosphamide once daily for 2 days is a most suitable method for the establishment of rabbit model of premature ovarian failure (POF).
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Objective To study the underlying mechanisms through which uric acid upregulates local renin-angiotensin system in adipocytes. Methods The primary cultured rat adipocytes were administered with 0, 1, 5, 10, and 15 mg/dl uric acid for 0, 12, 24, 48, and 72 h. Some of the pre-adipocytes were infected with siRNA-TLR2 or its negative control before differentiation. Then infected mature adipocytes were treated with 10 mg/dl uric acid for 48 h. After that procedure, mRNA levels of TLR2, TNF-α, IL-6, MCP-1, AGT, ACE1, AT1R, and AT2R were detected with real-time PCR method. The protein levels of TLR2 and NF-κB were detected by Western blotting. The concentrations of angiotensinⅡ( Ang Ⅱ) in the conditioned medium or cell lysate were measured using ELISA method. Results The mRNA levels mRNA of TLR2 increased in parallel with uric acid concentration. Moreover, it also increased with the time. By contrast, TLR2 mRNA expression decreased at 72 h. Uric acid increased levels of TNF-α, IL-6, and MCP-1 in adipocytes. It was also found that uric acid upregulated RAS components, including AGT, ACE1, AT1R, AT2R, and AngⅡ. However, siRNA-TLR2 infection significantly reduced the levels of TLR2 and NF-κB. As a result, both inflammatory cytokines and RAS components were significantly decreased in adipocytes. Conclusion Uric acid up-regulates RAS expression partially via TLR2 inflammatory signaling pathway in adipocytes.
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Objective To study the effect of gastrodin on arterial blood gas and brain injury of rats under simulated high altitude hypoxia environment. Methods A total of 60 adult healthy male Wistar rats were randomly divided into normal (N) group, hypoxia model (M) group, rhodiola crenulata (RC) group, low dose of gastrodin (GAS-L) group, medium dose of gastrodin (GAS-M) group and high dose of gastrodin (GAS-H) group (10 for each group). The intragastric administration on rats was continued for 7 days timely in each day. Under simulated 8000m altitude using low pressure oxygen cabin, the arterial blood gas of each group were tested, pathological changes of brain tissues were observed and related indexes of brain were detected after 12h hypoxia. Results Comparing with group N, the blood oxygen partial pressure (PO2), value of blood oxygen saturation (SO2), oxygenation index (PO2/FIO2), Na+ concentration (Na+), actual bicarbonate radical (HCO3–) significantly decreased (P<0.01), lactic acid (Lac), hemoglobin concentration (Hb) significantly increased (P<0.01) and pathological damage was inflicted in group M; and contents of malondialdehyde (MDA), hydrogen peroxide (H2O2) in brain tissue significantly increased (P<0.01), content of glutathione(GSH) and activity of glutathione peroxidase (GSH-Px) in brain tissue significantly decreased (P<0.01) in group M. Compared with group M, PO2, SO2 and PO2/FIO2 significantly increased (P<0.01, P<0.05) in group GAS-L; Na+ and HCO3– significantly increased (P<0.01, P<0.05) in three dose groups of GAS; Lac significantly decreased (P<0.01, P<0.05) in group GAS-L and GAS-H. Hb significantly increased (P<0.01) in group GAS-H, a rising trend appeared in group GAS-L but with no statistical significance. Damages of brain tissue were alleviated in group RC and three dose groups of GAS comparing with group M. Compared with group M, MDA significantly decreased (P<0.01) in three dose groups of GAS; there was a decreasing trend of H2O2 but with no statistical significance in three dose groups of GAS; GSH and GSH-Px significantly increased (P<0.01, P<0.05) in three dose groups of GAS. However, three groups of GAS has no dose dependent. Conclusion There was an protective effect of gastrodin on arterial blood gas and brain injury of rats under simulated high altitude hypoxia environment.
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BACKGROUND:The rabbit adipose-derived stem cells are mostly isolated by type I col agenase digestion, but rarely by explants culture method. OBJECTIVE:To isolate rabbit adipose-derived stem cells for adipogenic and osteogenic differentiation. METHODS:The rabbit adipose-derived stem cells were isolated from rabbit adipose by explants culture method, and cultured in vitro fol owed by morphological observation. The grow curve and cellsurface markers CD29, CD44, CD45 of passage 3 cells were analyzed respectively by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide and flow cytometry;cells from the third passages were induced for adipogenic and osteogenic differentiation by different revulsants, and cells were examined by oil red O staining and alizarin red staining . RESULTS AND CONCLUSION:The rabbit adipose-derived stem cells cultured in vitro exhibited a spindle-shaped appearance and could rapidly expand. Flow cytometry analysis revealed that the third passage of rabbit adipose-derived stem cells was positive for CD29, CD44, but negative for CD45. Rabbit adipose-derived stem cells were positive for oil red O staining at 14 days of adipogenic induction, and positive for alizarin red staining at 14 days of osteogenic induction. In conclusion, we could successful y isolate rabbit adipose-derived stem cells using explants culture method.
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Objective To study the feasibility and clinical significance of internal tourniquet (temporary arterial lumen blood flow occlusion by balloon catheter) for controlling the hemorrhage from traumatic neck and adjacent trunk arteries by temporarily occluding the arterial lumen blood flow.Methods The study involved 35 patients with traumatic neck and near trunk arteries who were firstly managed by internal tourniquet during operation to temporarily occlude the proximal aorta blood flow from May 1987 to February 2009.Each blocking time ranged from 30 to 70 minutes and the blocking was performed at an interval of 15 to 20 minutes.Then,surgical therapy was taken.Results After temporary proximal aorta blood flow occlusion with internal tourniquet,the operation presented few bleeding,with a clean operating field and clear anatomic structures.The total intraoperative blood loss was 100-400 ml.All patients were healed without ischemia of brains and limbs or relapse during the 3-14 years of followup.Conclusion Internal tourniquet,which can effectively reduce intraoperative blood loss and improve operation safety by temporarily occluding the proximal aorta blood flow,is an auxiliary approach for treating hemorrhage from traumatic neck and adjacent trunk arteries.
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<p><b>OBJECTIVE</b>To assess the effect of repeated gonadotropic stimulations on the developmental potential and growth differentiation factor-9 (GDF-9) expression of mouse oocytes.</p><p><b>METHODS</b>Female Kunming mice were treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG) for 3 times, and the control mice were treated with normal saline. The two groups of mice were both stimulated subsequently to obtain the mature oocytes. Immunocytochemical staining was employed to evaluate GDF-9 expression in the oocytes. The oocytes were then inseminated and cultured till the formation of blastocysts to compare the cleavage rate and blastocyst formation rate between the groups.</p><p><b>RESULTS</b>A total of 253 mature oocytes were obtained in the repeated stimulation group, with a mean of 11.5 oocytes from each mouse; 521 mature oocytes were obtained in the control group with a significantly greater mean number of 32.6 from each mouse (P<0.05). The average optical density and integrated optical density for GDF-9 expression were significantly lower in the oocytes in repeated stimulation group than in the control group (P<0.05 and 0.01, respectively). After insemination, the cleavage rate were comparable between repeated stimulation group and the control group (85.6% vs 88.8%), but the blastocyst formation rate was significantly lower in repeated stimulation group (20.8% vs 35.2%, P<0.01).</p><p><b>CONCLUSION</b>Repeated gonadal stimulation decreases the developmental potential of mouse oocytes possibly due to reduced GDF-9 expression.</p>
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Animais , Feminino , Camundongos , Células Cultivadas , Gonadotropinas , Farmacologia , Fator 9 de Diferenciação de Crescimento , Metabolismo , Oócitos , Biologia Celular , Metabolismo , Indução da Ovulação , MétodosRESUMO
OBJECTIVE: To prepare solid supersaturatable self-emulsifying drug delivery system of vinpocetine(VIN-S-sSEDDS) and to study its characteristics in vitro and in vivo.METHODS: VIN-S-sSEDDS was prepared by spray drying using hydroxypropyl methyl cellulose(HPMC) as supersaturation promoter and dextran-40 as solid carrier.VIN-S-sSEDDS was compared with conventional self-emulsifying drug delivery system containing vinpocetine(VIN-SEDDS) in respects of particle size,in vitro dissolution and bioavailability in rats(ig.).RESULTS: The particle size of VIN-S-sSEDDS(58.78 nm) was smaller than that of VIN-SEDDS(65.12 nm).The accumulative dissolution of VIN-S-sSEDDS within 2 h(88.7%) was higher than that of VIN-SEDDS(58.2%).The bioavailability of VIN-S-sSEDDS in rats(2.30) was higher than that of VIN-SEDDS(1.63).CONCLUSION: Prepared VIN-S-sSEDDS could apparently improve the dissolution and bioavailability of VIN,and it is superior to conventional self-emulsifying preparation.