Enhancing Erectile Function and Alleviating Andropause Symptoms: Clinical Efficacy of a Human Stem Cell Conditioned Medium Cream

Erectile dysfunction (ED) is increasingly prevalent in Japan, exceeding 30%, and increasing with age. Unhealthy lifestyle habits, obesity, insufficient exercise, and smoking have been implicated in its pathogenesis, along with endothelial dysfunction of the corpora cavernosa and impaired blood flow to the penis considered underlying factors. However, the current treatments are limited to Phosphodiesterase-5 (PDE5) inhibitors. ED is the primary symptom of andropathy. This study reports the clinical efficacy of human stem cell-conditioned medium cream for ED treatment. Ten men without underlying diseases suspected of andropause with ED (mean age 43.2 ± 4.4 y, Hb 15.2 ± 0.6 gm/dL, AST/ALT 30.2/37.9 ± 12.4/14.0, eGFR 82.7 ± 12.4 mL/min/1.73 m2) were targeted. The cream was applied twice daily to the genital and scrotal areas. The erectile hardness score (EHS), International Index of Erectile Function-5 (IIEF-5), and Aging Male Symptoms (AMS) scale were used to evaluate the participants before and 30 days after use, and the results were compared using paired t-tests. The post-use qualitative opinions were collected through interviews. Significant improvements were observed compared to baseline in the IIEF-5 (11.8 ± 4.6→17.2 ± 5.1, P < 0.001), and AMS (46.3 ± 6.7→37.6 ± 5.3, P < 0.001) scores post cream use. EHS did not show a statistically significant difference, but a trend towards improvement was observed. Qualitative feedback included increased morning erection, improved maintenance of erection during intercourse, and reduced post work fatigue. Human stem cell-conditioned medium contains endothelial growth factors that potentially contribute to the improvement of ED and andropause by enhancing corporal endothelial function. Future studies should include control groups to further investigate the efficacy of these treatments.

CONDITIONED MEDIUM OF HUMAN NASOPHARYNGEAL CARCINOMA EPITHELIOID CELL LINE CNE_(1) CONTAINED THE ACTIVITIES OF TRANSFORMED GROWTH-INHIBITING FACTORS

The hormone defined serum free conditioned medium (SFCM) of human nasopharyngeal carcinoma epithelioid cell line (CNE1) was assayed by both the 3H-thymidine incorporation test and the soft agar test. It was found that the SFCM stimulated the growth of long-term serum-free cultured CNE4 cells in ac-cordence with the fact that the growth rate of long-term serum-free cultured CNE1 cells was directly proportional to the plating density. Alternatively 5% SFCM inhibited the growth of short-term serum-free cultured CNE4 cells by 51% in which the indicator cell remained the responsiveness state of growing in the serum-supplemented medium to the effector of interest. Furthermore, SFCM resulted in the inhibition of anchorage-independent growth of CNE4 cells and A431 cells. Also in soft agar test. SFCM reduced the colony formation of NRK(?),9F cells in the presence of EGF or EGF plus TGF-β. These finding suggested that CNE4 secreted autocrine growth stimulating factor(s) and growth inhibiting factor(s) in the serum-free medium, the latter strongly reverse malignant phenotypes of CNE4 and A431 cells in serum-supplemented surrounding.

DAPT suppresses the proliferation of human glioma cell line SHG-44

Objective:To explore the suppressing effect ofγ-secretase inhibitor DAPT on proliferation of human glioma cell line SHG-44 in vitro and its mechanism.Methods:The SHG-44 cell was treated by DAPT with different concentration.The proliferation of cells was detected by MTT assay;cell cycle and TSC of CD133^+were determined by flow cytometry analysis technique;the key factor in Notch signaling pathway(Notch-1,Delta-1,Hes-1)was measured by reverse transcrip tase-polymerase chain reaction and western blotting.Results:DAPT inhibited the growth and proliferation of SHG-44 cells significantly(P<0.05).And the inhibiting effect on SHG-44 cells produced by DAPT showed a dose-dependent manner.DAPT increased the rate of cells in G_0/G_1 phase of SHG-44 cells,while it decreased the rate of cells in S phase.TSC of CD133^+was significantly reduced after DAPT treated SHC-44 cells.The expression of protein and mRNA of Notch-1,Delta-1 and Hes-1 were gradually downregulated with the increase of DAPT doses.Conclusions:DAPT can downregulate these key factor in Notch signaling pathway,reduce the TSC of CD133+and inhibit the proliferation of SHC-44 cells.

Effect of conditioned medium from neural stem cells on glioma progression and its protein expression profile analysis

BACKGROUND Emerging evidence suggests that the spread of glioma to the subventricular zone(SVZ)is closely related to glioma recurrence and patient survival.Neural stem cells(NSCs)are the main cell type in the SVZ region and exhibit tumor-homing ability.AIM To evaluate the effects of conditioned medium(CM)derived from SVZ NSCs on the cancer-related behaviors of glioma cells.METHODS The characteristics of SVZ hNSCs were identified by immunofluorescence.The normoxic-hNSC-CM and hypoxic-hNSC-CM(3%O2,oxygen-glucose deprived[OGD]culturing)were collected from 80%-90%confluent SVZ NSCs in sterile conditions.The CCK8 and Transwell assays were used to compare and evaluate the effects of normoxic-CM and hypoxic-CM on glioma proliferation and invasion.Then proteins secreted from SVZ NSCs into the CM were investigated by mass spectrometry,and the potential effects of candidate protein NCAN in the regulation of glioma progression were examined by CCK8 and Transwell assays.RESULTS The CM from SVZ NSCs significantly increased the proliferation and invasion of glioma cells,particularly the CM from OGD NSCs induced under hypoxic conditions.Furthermore,the secreted protein neurocan(NCAN)in CM from OGD NSCs was identified by proteomic analysis.NCAN was expressed in glioma cells and played regulatory roles in mediating the progression of glioma cells mainly via the Rho/Rho-associated protein kinase pathway.CONCLUSION Our study identified a potential interactive mechanism between SVZ NSCs and glioma cells,in which SVZ NSCs promote glioma progression via the secreted protein NCAN.These findings suggested that exploring the CM derived from cells could be a novel strategy for optimizing treatments and that NCAN derived from SVZ NSCs may be a potential new target in glioma progression.

THE STUDIES ON MONOCLONAL ANTIBODY SZ-39 AGAINST HUMAN GLIOMA CELLS

A hybridoma cell line SZ-39 secreting monoclonal antibody against the human glioma cell has been established by a fusion between NS-1 myeloma cells and spleen cells from mice immunized with human glioma cell lines. Monoclonal antibody (McAb) SZ-39 was analyzed by ELISA, quantitative absorption, indirect immunofluorescence and ABC immunohistology. McAb SZ-39 strongly bound to 9/10 glioma cell lines, 17/20 glioma tissues, weakly bound to one liver cancer cell line and 1/2 lung cancer line, but they did not band with other tested human cancer linse. NcAb SZ-39 have no cross-reaction with lymphocyte, ABC red blood cells, white blood cells, blood platelet, normal bone marrow cells, fibroblast cells and 12 normal human tissues.The result indicated the antigen recognized by McAb SZ-39 may be a glioma-associated antigen <GAA). This GAA was analyzed by means of Western blotting. It was a MW 180 Kd glycopro-tein. The 131I-McAb SZ-39 specifically localized in human glioma xenografted in nude mice that indicate it may be useful in radioimmunoimaging and as a target for immunotherapy on human glioma.

Adiponectin Supports Human Glioma Cells Survival against Temozolomide through Enhancement of Autophagic Response in Glioma Cells

Objective: To investigate the role of adiponectin in human glioma cell lines against the temozolomide and the molecular regulation mechanism. Methods: Human glioma cell lines U251 and U-87MG were cultured in Dulbecco’s modified eagle medium (DMEM) containing 4500 mg/L glucose. MTT was used to measure cell growth ratio. Western blot was used to detect the protein levels of autophagy-related protein (Beclin 1, LC3 I/II, p62) and phosphorylated AMPK (p-AMPK) in human glioma cell lines. After AICAR and Compound C were administered, the change of p-AMPK and the autophagy level were examined by western blot. Results: While adiponectin stimulates AMPK in phosphatase and up-regulates the level of autophagy, human glioma cell lines obtain more resistance against the temozolomide, which is facilitated by AICAR and weakened by Compound C. Conclusion: As an important adipokine, adiponectin can up-regulate the glioma cell autophagy by activating the AMPK signaling pathway which increases the resistance of glioma cells to temozolomide.

Human umbilical cord mesenchymal stem cells promote peripheral nerve repair via paracrine mechanisms

Human umbilical cord-derived mesenchymal stem cells （hUCMSCs） represent a promising young-state stem cell source for cell-based therapy. hUCMSC transplantation into the transected sciatic nerve promotes axonal regeneration and functional recovery. To further clarify the para-crine effects of hUCMSCs on nerve regeneration, we performed human cytokine antibody array analysis, which revealed that hUCMSCs express 14 important neurotrophic factors. Enzyme-linked immunosorbent assay and immunohistochemistry showed that brain-derived neurotrophic factor, glial-derived neurotrophic factor, hepatocyte growth factor, neurotrophin-3, basic fibroblast growth factor, type I collagen, fibronectin and laminin were highly expressed. Treatment with hUCMSC-conditioned medium enhanced Schwann cell viability and proliferation, increased nerve growth factor and brain-derived neurotrophic factor expression in Schwann cells, and enhanced neurite growth from dorsal root ganglion explants. These ifndings suggest that paracrine action may be a key mechanism underlying the effects of hUCMSCs in peripheral nerve repair.

Low level of activin A secreted by fibroblast feeder cells accelerates early stage differentiation of retinal pigment epithelial cells from human pluripotent stem cells

Human pluripotent stem cells (hPSC) differentiated to retinal pigment epithelial cells (RPE) provide a promising tool for cell replacement therapies of retinal degenerative diseases. The in vitro differentiation of hPSC-RPE is still poorly understood and current differentiation protocols rely on spontaneous differentiation on fibroblast feeder cells or as floating cell aggregates in suspension. The fibroblast feeder cells may have an inductive effect on the hPSC-RPE differentiation, providing variable signals mimicking the extraocular mesenchyme that directs the differentiation in vivo. The effect of the commonly used fibroblast feeder cells on the hPSCRPE differentiation was studied by comparing suspension differentiation in standard RPEbasic (no bFGF) medium to RPEbasic medium conditioned with mouse embryonic (mEF-CM) and human foreskin (hFF-CM) fibroblast feeder cells. The fibroblast secreted factors were found to enhance early hPSC-RPE differentiation. The onset of pigmentation was faster in the conditioned media (CM) compared to RPEbasic for both human embryonic (hESC) and induced pluripotent (iPSC) stem cells, with the first pigments appearing around two weeks of differentiation. After four weeks of differentiation, CM conditions consistently contained higher number of pigmented cell aggregates. The ratio of PAX6 and MITF positive cells was quantified to be clearly higher in the CM conditions, with mEFCM containing most positive cells. The mEF cells were found to secrete low levels of activin A growth factor that is known to regulate eye field differentiation. As RPEbasic was supplemented with corresponding, low level (10 ng/ml) of recombinant human activin A, a clear increase in the hPSC-RPE differentiation was achieved. Thus, inductive effect provided by feeder cells was at least partially driven by activin A and could be substituted with a low level of recombinant growth factor in contrasts to previously reported much higher concentrations.

人骨髓间充质干细胞通过YAP影响人脂肪肉瘤SW872细胞的生物学行为

目的:观察人骨髓间充质干细胞(hMSCs)条件培养基(CM)与人脂肪肉瘤SW872细胞共培养后对肿瘤细胞增殖和迁移能力的影响,探讨hMSCs CM对脂肪肉瘤细胞的作用及可能的作用机制。方法:体外培养hMSCs,采用慢病毒方法分别转染慢病毒空载体shNS(对照组)和慢病毒shRNA Yes相关蛋白(YAP)(shYAP-hMSCs组),采用实时荧光定量PCR(RT-qPCR)法和Western blotting法检测各组hMSCs中YAP mRNA和蛋白表达水平,提取CM。体外培养SW872细胞,分为对照组(正常培养)、hMSCs CM组和shYAP-hMSCs CM组。采用CCK-8法检测各组细胞增殖活性,流式细胞术检测各组细胞凋亡率,细胞划痕实验检测各组细胞划痕愈合率,Western blotting法检测各组细胞中YAP、基质金属蛋白酶9(MMP-9)和细胞周期蛋白D1(cyclin D1)蛋白表达水平。结果:与对照组比较,shYAP-hMSCs组hMSCs中YAP mRNA和蛋白表达水平降低(P<0.01),表明成功构建了shYAP-hMSCs稳定转染细胞株。CCK-8法,与对照组比较,hMSCs CM组SW872细胞增殖活性升高(P<0.05),shYAP-hMSCs CM组SW872细胞增殖活性降低(P<0.01);流式细胞术,与对照组比较,hMSCs CM组SW872细胞凋亡率无明显变化(P>0.05),shYAP-hMSCs CM组SW872细胞凋亡率升高(P<0.01);细胞划痕实验,与对照组比较,hMSCs CM组SW872细胞划痕愈合率升高(P<0.05),shYAP-hMSCs CM组SW872细胞划痕愈合率降低(P<0.01);Western blotting法,与对照组比较,hMSCs CM组SW872细胞中YAP、MMP-9和cyclin D1蛋白表达水平差异无统计学意义(P>0.05),shYAP-hMSCs组SW872细胞中YAP、MMP-9和cyclin D1蛋白表达水平降低(P<0.05或P<0.01)。结论:hMSCs参与调控人脂肪肉瘤SW872细胞增殖和迁移,其机制可能与YAP表达有关。

乳酸干预破骨细胞条件培养基促进内皮细胞血管的生成

背景:聚乳酸作为可降解的骨组织工程支架材料被广泛用于组织再生与修复研究,在促进组织愈合与新骨形成、血管生成中具有重要作用。目的:观察聚乳酸降解终产物乳酸对破骨细胞的作用,以及乳酸干预后破骨细胞条件培养基对血管内皮细胞增殖、迁移和小管形成功能的影响。方法:(1)取对数生长期的小鼠单核巨噬细胞系RAW264.7,贴壁后分别加入含0,5,10,20 mmol/L乳酸的破骨诱导培养基(含核因子κB受体活化因子配体、体积分数10%胎牛血清的DMEM培养基),培养5 d后分别进行抗酒石酸酸性磷酸酶与细胞骨架纤维状肌动蛋白染色,培养24h后采用RT-PCR检测抗酒石酸酸性磷酸酶5 m RNA表达。(2)取对数生长期的RAW264.7细胞,贴壁后分2组培养:对照组加入破骨诱导培养基,实验组加入含10 mmol/L乳酸的破骨诱导培养基,培养5 d后更换为无血清DMEM培养基继续培养24 h,离心取上清液,分别与等体积含体积分数10%胎牛血清DMEM培养基混合后作用条件培养基备用。取对数生长期的人脐静脉内皮细胞,分别与对照组、实验组条件培养基共培养,通过CCK-8、Transwell、划痕、成管实验观察细胞的增殖、迁移和成管能力,通过RT-PCR和Western Blot检测血管生成相关基因和蛋白的表达。结果与结论:(1)抗酒石酸酸性磷酸酶与肌动蛋白染色结果显示,5,10 mmol/L乳酸可促进RAW264.7细胞破骨向分化,其中以10 mmol/L乳酸的促进作用更显著;RT-PCR检测结果显示,5,10,20 mmol/L乳酸均可提高酒石酸酸性磷酸酶5 m RNA的表达,其中以10 mmol/L乳酸的提高作用最显著;(2)与对照组条件培养基相比,实验组条件培养基可促进人脐静脉内皮细胞的增殖、迁移与成管能力(P<0.05),提高血管内皮生长因子、血管生成素1的m RNA和蛋白表达(P<0.05);(3)结果表明,乳酸诱导分化的破骨细胞条件培养基可促进内皮细胞的血管生成,机制可能与提高血管内皮生长因子、血管生成素1表达相关。

年轻化间充质干细胞条件培养基促进小鼠创面愈合的研究

背景间充质干细胞(mesenchymal stem cell,MSC)条件培养基无细胞疗法是创伤修复领域的一种新型治疗方案,但其治疗效果受间充质干细胞状态的影响。目的探究年轻化MSC培养基对小鼠创面愈合的作用。方法建立间充质干细胞复制性衰老(传代至15~20代)模型,检测其衰老指征;使用小分子化合物[丙戊酸(valproic acid,VPA)和RepSox]逆转衰老间充质干细胞并验证逆衰老效果。分别收集衰老MSC培养基与年轻化MSC培养基。用收集到的两种条件培养基培养人成纤维细胞,使用CCK-8实验、Ki-67免疫荧光、划痕实验和Transwell实验检测这两种条件培养基对成纤维细胞增殖、迁移和侵袭能力的影响。分别用衰老MSC培养基、年轻化MSC培养基和0.9%氯化钠注射液治疗C57BL/6小鼠背部皮肤全层缺损模型,评估3种治疗方法对创面愈合的影响。结果与衰老的间充质干细胞相比,经小分子处理后衰老间充质干细胞SA-β-gal阳性率降低(P<0.01),且免疫荧光检测显示其衰老基因P21、P16表达降低(P<0.01)。经年轻化MSC培养基处理的成纤维细胞增殖、迁移和侵袭能力增强(P<0.01)。创伤模型显示,与对照组相比,年轻化MSC培养基治疗组小鼠创面愈合速度加快(P<0.01);年轻化MSC培养基治疗组小鼠创面HE染色显示表皮细胞再上皮化加快(P<0.01)、Masson染色显示创面胶原沉积量增加(P<0.01)、CD31免疫荧光染色显示创面血管量增加(P<0.01)。结论年轻化MSC培养基对小鼠创面修复或有促进作用。

人脐带间充质干细胞条件培养基及外泌体对肝癌细胞增殖、迁移、侵袭和凋亡的影响

背景:间充质干细胞可通过分泌含细胞因子、生长因子和外泌体的细胞外囊泡调节肿瘤微环境,对肿瘤细胞生物学行为进行精准调控。目的:研究人脐带间充质干细胞条件培养基及外泌体对肝癌细胞生物学特性的影响。方法:收集人脐带间充质干细胞培养上清,通过高速离心并过滤获得人脐带间充质干细胞条件培养基;收集人脐带间充质干细胞培养上清,通过超高速梯度离心法提取人脐带间充质干细胞外泌体。使用PKH26标记人脐带间充质干细胞外泌体,与肝癌细胞MHCC97-H共培养,荧光显微镜观察MHCC97-H细胞摄取外泌体情况。通过CCK-8增殖实验、Transwell迁移和侵袭实验以及流式凋亡实验评估人脐带间充质干细胞条件培养基、人脐带间充质干细胞外泌体对肝癌细胞生物学功能的影响。结果与结论:(1)人脐带间充质干细胞外泌体可被MHCC97-H细胞摄取且主要分布于细胞质中;(2)人脐带间充质干细胞条件培养基处理后,MHCC97-H细胞的增殖、迁移和侵袭能力显著增强(P<0.001,P<0.05,P<0.01),凋亡能力显著下降(P<0.001);而人脐带间充质干细胞外泌体处理后,MHCC97-H细胞的增殖能力下降(P<0.001),迁移、侵袭及凋亡能力显著增强(P<0.001);(3)结果表明,人脐带间充质干细胞条件培养基具有促进MHCC97-H细胞增殖、迁移、侵袭和抑制凋亡的能力;而人脐带间充质干细胞外泌体则具有促进MHCC97-H细胞迁移、侵袭及凋亡和抑制增殖的能力。

淫羊藿苷预处理增强人牙周膜干细胞对M1型巨噬细胞的影响

背景:人牙周膜干细胞对M1型巨噬细胞促炎功能有一定抑制作用,具有抗炎等药理活性的淫羊藿苷是否能增强人牙周膜干细胞对M1型巨噬细胞的抑制作用尚不明确。目的:探究淫羊藿苷预处理人牙周膜干细胞后对M1型巨噬细胞的影响。方法:分离培养原代人牙周膜干细胞,并进行鉴定。对THP-1细胞进行诱导,采用免疫荧光染色及PCR进行M1型巨噬细胞鉴定。用含浓度10^(-7),10^(-6),10^(-5),10^(-4)mol/L淫羊藿苷的α-MEM完全培养基培养人牙周膜干细胞1,3,5,7 d,采用CCK-8法筛选合适的淫羊藿苷浓度进行实验。将α-MEM完全培养基、未处理的人牙周膜干细胞α-MEM条件培养基及淫羊藿苷预处理24 h的人牙周膜干细胞α-MEM条件培养基与RPMI-1640完全培养基以1∶1的比例对M1型巨噬细胞进行条件培养,分别为对照组、未处理组及预处理组,24 h后RT-PCR法检测M1型巨噬细胞炎症因子的mRNA表达情况;ELISA法检测M1型巨噬细胞炎症因子的蛋白表达情况;Western blot检测M1/M2型巨噬细胞表面标记物及核转录因子κB通路相关蛋白的表达。结果与结论:(1)CCK-8检测结果显示,10^(-7),10^(-6),10^(-5),10^(-4)mol/L淫羊藿苷对人牙周膜干细胞均无细胞毒性,且从第5天起,各浓度都提高了细胞活力,促进细胞增殖,选择10^(-4)mol/L淫羊藿苷进行后续实验。(2)RT-PCR法及ELISA检测结果显示,与对照组相比,未处理组及预处理组均降低了M1型巨噬细胞白细胞介素1β、白细胞介素6及肿瘤坏死因子α的表达与分泌(P<0.05),且预处理组低于未处理组(P<0.05)。(3)Western blot检测结果显示,与未处理组相比,预处理组CD86的表达明显降低(P<0.05);与对照组相比,未处理组和预处理组M2型巨噬细胞表面标志物CD206的表达均升高(P<0.01),且预处理组明显高于未处理组(P<0.01);M1型巨噬细胞经24 h条件培养后,与对照组相比,核转录因子κB/P65的表达在未处理组和预处理组均有降低(P<0.01),p-IκBα的表达仅在预处理组降低(P<0.01);与未处理组相比,预处理组核转录因子κB/P65和p-IκBα的表达均显著降低(P<0.05),而IκBα在3组中的表达差异无显著性意义。(4)上述结果证实,淫羊藿苷增强了人牙周膜干细胞对M1型巨噬细胞的抑制作用,此作用可能与抑制了巨噬细胞的核转录因子κB信号通路相关。

不同来源条件培养基对人牙髓干细胞增殖的影响

背景:利用条件培养基与人牙髓干细胞共培养,初步探索可以快速促进人牙髓干细胞增殖的方法,为今后细胞治疗、扩增高质量种子细胞提供研究基础。目的:初步探究不同来源条件培养基对人牙髓干细胞增殖的影响。方法:体外分离培养人牙髓干细胞,并用流式细胞术鉴定。然后将胎牛血清超高速低温离心产物、骨折患者尿液超高速低温离心产物、骨髓来源树突状细胞体外培养上清液、人皮肤成纤维细胞培养上清液与低糖DMEM培养液配置成条件培养基后,再分别与人牙髓干细胞共培养,使用细胞无标记培养观察装置(BioStation-T)连续拍摄各组细胞生长72 h的照片、使用实时无标记细胞分析仪(RTCA)动态监测150 h,比较各组细胞的标准化细胞指数值的差异。结果与结论:①人牙髓干细胞为典型的间充质干细胞形态,表达间充质干细胞表面标志物CD90和CD105,不表达CD34和CD45;②细胞无标记培养观察装置动态监测时,发现胎牛血清超高速低温离心组的颗粒物质被吸收时间要显著早于其他实验组和空白对照组;③在实时无标记细胞分析仪检测时,与空白对照组相比,胎牛血清超高速低温离心组、骨髓来源树突状细胞体外培养上清液组、人皮肤成纤维细胞培养上清液组的标准化细胞指数值均显著升高(P<0.001),骨折患者尿液超高速低温离心组的标准化细胞指数值显著降低(P<0.001);④结果表明,胎牛血清超高速低温离心产物、骨髓来源树突状细胞体外培养上清液、人皮肤成纤维细胞培养上清液所配置的条件培养基能促进人牙髓干细胞增殖,其中胎牛血清超高速低温离心产物所配置的条件培养基对细胞还有一定保护作用。

多发性骨髓瘤细胞条件培养液对人脐带间充质干细胞增殖和分化的影响

背景:近年来,在肿瘤微环境中干细胞与肿瘤的相互作用已成为研究热点,但是多发性骨髓瘤细胞条件培养液对人脐带间充质干细胞影响的实验鲜有报道。目的:探讨多发性骨髓瘤细胞条件培养液对人脐带间充质干细胞增殖、迁移及分化能力的影响。方法:以组织块贴壁法提取人脐带间充质干细胞,同时培养并提取人多发性骨髓瘤RPMI8226和U266细胞上清液制备条件培养液。按培养基的不同进行细胞分组:对照组人脐带间充质干细胞仅用L-DMEM完全培养液培养;实验组人脐带间充质干细胞用20%RPMI8226细胞条件培养液或20%U266细胞条件培养液和80%L-DMEM完全培养液培养。培养24 h,通过CCK-8、EDU、结晶紫染色观察细胞增殖情况,划痕法评估细胞迁移情况,成骨成脂诱导实验检测细胞分化能力,Real-time PCR检测细胞中周期蛋白基因p16、p21 mRNA表达。结果与结论:实验组细胞的增殖数量多于对照组(P <0.05),迁移率高于对照组(P <0.05),矿化结节染色面积小于对照组(P <0.05),脂滴阳性染色面积大于对照组(P <0.05);实验组的细胞周期基因p16、p21 mRNA表达低于对照组(P <0.05)。结果表明,人多发性骨髓瘤细胞条件培养液可促进人脐带间充质干细胞的增殖、迁移和成脂分化,抑制成骨分化,可能与其抑制p16、p21基因表达有关。

siRNA沉默钙蛋白酶1对人脑胶质瘤细胞LN229增殖及侵袭的影响

目的 探讨siRNA沉默钙蛋白酶1(CAPN1)对人脑胶质瘤细胞LN229增殖及侵袭的影响。方法 取人脑胶质瘤细胞LN229于37℃水浴中解冻后制备单细胞悬液,接种于细胞培养板中,融合度至90%左右,将siRNA系列、CAPN1 siRNA序列分别转染细胞,分为实验组(CAPN1 siRNA)与siRNA组,以未转染的细胞作为对照组。采用实时荧光定量PCR(qRT-PCR)测定LN229细胞中CAPN1 mRNA表达;采用MTT法测定LN229细胞增殖情况;采用Transwell小室测定LN229细胞侵袭及迁移情况;采用Western blot测定LN229细胞Bax、Bcl-2和CAPN1蛋白表达。结果 与对照组和siRNA组比较,实验组CAPN1 mRNA表达明显降低(P<0.05);与对照组和siRNA组比较,实验组LN229细胞增殖率明显降低(P<0.05);与对照组和siRNA组比较,实验组LN229细胞侵袭率明显降低(P<0.05);与对照组和siRNA组比较,实验组LN229细胞迁移数目明显降低(P<0.05);与对照组和siRNA组比较,实验组LN229细胞Bax蛋白表达灰度值明显升高,而Bcl-2和CAPN1蛋白表达灰度值明显降低(P<0.05)。而对照组和siRNA组CAPN1 mRNA表达、LN229细胞增殖率、LN229细胞侵袭率、LN229细胞迁移数目、Bax、Bcl-2和CAPN1蛋白表达灰度值比较差异无统计学意义(P>0.05)。结论 siRNA沉默CAPN1可抑制人脑胶质瘤细胞LN229增殖,抑制脑胶质瘤细胞侵袭和迁移,上调Bax表达及下调Bcl-2和CAPN1蛋白表达。

5个品系小鼠胚胎干细胞系建立的方法学比较(英文)

以 70 %的大鼠心脏细胞条件培养基 (RH CM)为培养液 ,以小鼠胚胎成纤维细胞 (PMEF)为饲养层 ,采用添加 1%鸡血清的消化液和“连续离散法”作为小鼠ES细胞建系的改进方法 ,比较了 5个品系小鼠ES细胞系建立的特点。与常规方法相比 ,3个近交系小鼠 12 9 ter、C5 7BL 6J、BALB c的ES细胞建系率分别由 11 8%、3 7%和 2 9%提高到 33 3%、13 3%和 19 4 % ,差异十分显著 ;直接采用改进的方法建立KM和ICR小鼠ES细胞系 ,建系率分别达 12 %和 4 2 1%。讨论了ICM增殖的时间 ,即离散时机对ES集落形成及建系率的影响 ,结果显示 :12 9 ter、C5 7BL 6J、BALB c、KM和ICR小鼠品系ICM适宜的离散时机分别为增殖 4～ 6d、3～ 3 5d、4d、4～ 5d和 4～ 5d ;同时 ,讨论了不同ES细胞建系所需最适宜的消化液浓度 ,其中BALB c小鼠的ES细胞对高浓度的消化液十分敏感 ,0 0 5 %Trypsin 0 0 0 8%EDTA是其比较理想的离散浓度。设计了两种离散方法 ,即“一次离散法”和“连续离散法” ,用来离散增殖的ICM和ICM离散后出现的ES集落 ,结果表明 :后者在建系过程中的作用明显优于前者。RH CM与添加LIF的常规ES细胞培养基相比 ,不但具有显著抑制小鼠ES细胞分化、维持其二倍体核型的作用 ,而且明显促进ES细胞的贴壁生长。

CpG ODN107增强人脑胶质瘤细胞放射敏感性的研究

目的:探讨CpG ODN107增加人脑胶质瘤细胞CHG-5放射敏感性的作用及可能机制。方法:用不同浓度CpG ODN107处理CHG-5细胞,然后经β射线照射,MTT法检测细胞的增殖情况,体外培养集落形成率与细胞划痕法观察细胞的集落形成和迁移能力,ELISA法检测细胞TNF-α的分泌水平。结果:CpG ODN107抑制CHG-5细胞的增殖,并呈量效关系。CpG ODN107(10μg/ml)联合β射线照射,抑制CHG-5细胞的增殖作用最强。CpG ODN107(10μg/ml)同样能抑制CHG-5细胞增殖和迁移,并且增加了TNF-α的分泌,在联合β射线照射后作用更加显著。结论:CpG ODN107对CHG-5有明显的放射增敏作用,其机制可能与CpG ODN107增强肿瘤细胞TNF-α的分泌有关。

悬浮法培养C6胶质瘤细胞系和该细胞系中脑肿瘤干细胞的分离

目的研究应用无血清培养基悬浮法培养C6胶质瘤细胞系的方法;并分离该细胞系中的脑肿瘤干细胞,观察其生长方式和分化特征。方法应用培养大鼠神经干细胞的培养基和培养方法,在无血清培养基中采用悬浮法培养大鼠C6胶质瘤细胞系;再将分离获得的悬浮生长的脑肿瘤干细胞接种于含血清培养基,观察其分化;应用有限稀释和单克隆形成实验确定该细胞系中肿瘤干细胞的比例。结果C6胶质瘤细胞系中有约1.18%的肿瘤细胞在无血清培养基中能够存活、增殖,形成自由漂浮的细胞球;这种细胞球具有人脑肿瘤干细胞球的特征,并可连续传代,若重新接种于含血清培养基中可重新贴壁分化,贴壁分化后细胞形态与直接在含血清培养基中培养的C6胶质瘤细胞系无明显差别。结论C6大鼠胶质瘤细胞系中含有比例较低的、具有增殖和分化能力脑肿瘤干细胞(约1.18%),该细胞系可在含生长因子的无血清培养基中悬浮生长,并维持细胞系。

人毛乳头细胞条件培养基对毛乳头细胞的作用

目的:研究人毛乳头细胞条件培养基对高传代人毛乳头细胞的生物作用。方法:通过体外培养低传代的人毛乳头细胞,收集其上清配制成条件培养基,用此条件培养基培养高传代人毛乳头细胞,观察其细胞的3H-TdR计数值及细胞超微结构的变化。结果:用低传代人毛乳头细胞上清液培养过的高传代人毛乳头细胞3H-TdR计数值明显高于对照组(P<0.01)。在电镜下可见高传代人毛乳头细胞核糖体丰富、内质网分泌成池。结论:低传代人毛乳头细胞条件培养基有明显促进高传代人毛乳头细胞活性的作用。