犬阴道上皮细胞和平滑肌细胞可在体外长期稳定培养

背景：体外分离培养获得足够活性良好的种子细胞是构建阴道组织工程的关键。文献报道阴道上皮细胞体外纯化培养和传代较为困难,尤其是体外长期培养犬等大动物的阴道种子细胞尚未见报道。 目的：建立体外稳定培养犬阴道上皮细胞和平滑肌细胞方法。 方法：获取犬小块阴道组织,机械分离阴道黏膜上皮,Dispase酶和胰蛋白酶分步消化收集上皮细胞,接种于无血清角化细胞培养液中培养和传代;机械分离阴道平滑肌组织后采用Ⅱ型胶原酶消化获得平滑肌细胞,在含体积分数10%胎牛血清的DMEM培养液中连续培养传代。动态观察上皮细胞和平滑肌细胞生长增殖情况,分别采用特异性抗体行细胞免疫化学染色鉴定。 结果与结论：原代培养的上皮细胞24-36 h后开始贴壁铺展,四五天后呈对数生长,七八天可达70%融合,为单一的上皮细胞,呈典型铺路石样,未见成纤维细胞混杂。每四五天可传代1次,连续传代六七次,细胞免疫化学染色角蛋白AEl/AE3抗体阳性。平滑肌细胞原代培养24 h后贴壁呈梭形,此后呈对数生长,4 d后融合呈典型的“峰和谷”样,每三四天可传代1次,连续传代七八次,细胞免疫化学染色示α-肌动蛋白染色阳性。结果证实,犬阴道上皮细胞和平滑肌细胞可在体外长期稳定培养,可为体外构建组织工程化阴道提供足够的种子细胞。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

雌激素增强失血性休克大鼠肠系膜淋巴管的收缩功能

目的:观察17β-雌二醇(E2)对失血性休克大鼠肠系膜淋巴微循环和离体肠系膜淋巴管收缩性的作用及其与淋巴管平滑肌细胞(LSMCs)内外钙离子浓度([Ca2+])差的关系.方法:雄性Wistar大鼠随机均分为假手术组、休克组和休克+E2组,建立失血性休克模型[(40±2)mmHg维持1.5 h,液体复苏],休克+E2组在...     

大鼠远端肺动脉平滑肌细胞分离与原代培养

目的探索简便、高效原代培养大鼠远端肺动脉平滑肌细胞的方法,为研究肺动脉高压发病机制提供实验材料。方法采用显微操作和分次酶消化法进行原代培养并与传统酶消化法比较。对培养的细胞进行形态学观察、平滑肌α-actin免疫荧光细胞化学法鉴定、激光扫描共聚焦显微镜计算纯度。结果两种酶消化法均可获得高纯度的远端肺动脉平滑肌细胞。镜下细胞呈典型的"峰-谷"状生长,胞质特异的α-actin阳性表达,细胞纯度达98%。分次酶消化法获得的细胞数及细胞存活率均大于传统酶消化法,并且提前了3d得到足够进行实验的细胞数量。结论本法简便、可靠、低成本,短期内可获得大量高纯度、功能良好的远端肺动脉平滑肌细胞。     

大鼠远端肺动脉平滑肌细胞分离与原代培养

目的 探索简便、高效原代培养大鼠远端肺动脉平滑肌细胞的方法,为研究肺动脉高压发病机制提供实验材料.方法 采用显微操作和分次酶消化法进行原代培养并与传统酶消化法比较.对培养的细胞进行形态学观察、平滑肌α-actin免疫荧光细胞化学法鉴定、激光扫描共聚焦显微镜计算纯度.结果 两种酶消化法均可获得高纯度的远端肺动脉平滑肌细胞.镜下细胞呈典型的"峰-谷"状生长,胞质特异的α-actin阳性表达,细胞纯度达98%.分次酶消化法获得的细胞数及细胞存活率均大于传统酶消化法,并且提前了3 d得到足够进行实验的细胞数量.结论 本法简便、可靠、低成本,短期内可获得大量高纯度、功能良好的远端肺动脉平滑肌细胞.     

小肠黏膜下层无细胞基质作为阴道平滑肌细胞载体的可行性

背景:目前国内外用于阴道组织工程研究的主要支架材料聚乙醇酸存在降解过快等缺陷。天然脱细胞支架材料尤其是小肠黏膜下层逐渐成为组织工程研究的重点。目的:探索用猪小肠黏膜下层基质作为组织工程学阴道细胞载体的可行性。方法:取新西兰雌兔,分离出阴道平滑肌组织块,组织块+酶消化法原代培养阴道平滑肌细胞。体外培养传代后作为种子细胞接种于自制猪小肠黏膜下层基质体外联合培养,倒置显微镜动态观察细胞形态及生长增殖情况,分别于1,2,3,4周时取标本,行组织学检查。结果与结论:①体外成功培养出阴道平滑肌细胞,倒置显微镜下,见培养的阴道平滑肌细胞呈现长梭状,细胞集结于培养皿上形成典型的"峰和谷"样构型。②黏膜下层无细胞基质外观呈白色,半透明,有一定韧性。苏木精-伊红染色未见细胞成分存在。③阴道平滑肌细胞-肠黏膜下层标本切片苏木精-伊红染色后,光镜下可见细胞成分逐渐增多,由表浅向深层部位生长。④阴道平滑肌细胞-肠黏膜下层标本切片采用抗兔平滑肌α-肌动蛋白单克隆抗体免疫组化染色后,均可见抗兔α-Actin的阳性细胞。结果初步证明了猪小肠黏膜下层基质可作为一种平滑肌细胞载体。     

平滑肌细胞对共培养体系滋养细胞侵袭力与MMP-2及MMP-9表达的影响

目的模拟体内环境,建立可保持平滑肌细胞与绒毛外细胞滋养层细胞(EVCT)生物学特性的共培养细胞模型,应用于研究平滑肌细胞与滋养细胞理化特性与滋养细胞的侵袭行为。方法利用组织块培养法培养脐动脉平滑肌细胞,组织块培养、胰酶消化和Percoll梯度沉降,收集纯化人早孕绒毛组织的滋养细胞,免疫组化检测细胞的纯度。将滋养细胞与平滑肌细胞分别放入Transwell的上下小室,观察该模型下滋养细胞形态变化、细胞活力、侵袭力改变与分泌功能等特性。结果免疫组化显示EVCT的细胞角蛋白7阳性表达的细胞数占95%以上,SMC a-actin阳性表达的细胞数也超过95%,证实共培养系统中EVCT和SMC纯度均在95%以上,且生物学特性得以维持。上室中的EVCT保持了其侵袭能力,且平滑肌细胞能促进滋养细胞增殖活性、侵蚀能力及MMP2、MMP9的表达。结论成功地建立了平滑肌细胞与滋养细胞原代共培养系统模型,便于研究滋养细胞侵袭和子宫螺旋动脉重铸障碍的分子机制。     

三种方法培养慢性阻塞性肺疾病大鼠气道平滑肌细胞的比较

背景：原代培养方法被广泛应用于正常和哮喘大鼠气道平滑肌细胞的培养,但少见用于慢性阻塞性肺疾病大鼠气道平滑肌细胞培养的报道。 目的：建立大鼠慢性阻塞性肺疾病模型,比较组织块法、酶消化法和改良组织块消化法原代培养模型大鼠气道平滑肌细胞生长情况的差异。 方法：将16只清洁级雄性健康SD大鼠随机分成2组,对照组正常饲养,慢性阻塞性肺疾病组用熏烟法建立大鼠慢性阻塞性肺疾病模型,显微镜下观察大鼠肺组织病理学特点。分别应用上述3种方法原代培养慢性阻塞性肺疾病大鼠气道平滑肌细胞,相差显微镜下观察细胞形态,用α-平滑肌肌动蛋白免疫组织化学染色鉴定细胞类型。 结果与结论：经病理学证实成功构建慢性阻塞性肺疾病大鼠模型。在倒置相差显微镜下见培养的细胞表现为典型的 “谷-峰状”生长。经免疫细胞化学染色后,可见胞质呈棕色阳性反应,所培养的细胞有94%以上为气道平滑肌细胞。3种方法在耗时和细胞质量方面均无明显差别,但组织块法更经济、稳定可靠和简单。     

淋巴管壁、血管壁氧耗量的测量

应用OBN—Ⅲ型生物组织氧耗仪,测量了大鼠新鲜淋巴管壁、腔静脉管壁、腹主动脉管壁1min的氧耗量。结果征明:15只大鼠腹主动脉壁内膜面氧耗量明显高于淋巴管壁和腔静脉管壁内膜面氧耗量(P<0.001);淋巴管壁氧耗量高于腔静脉管壁(P<0.001);而腔静脉管壁氧耗量最低。结果说明腹主动脉管壁氧耗量高与它在执行功能过程中内皮细胞与平滑肌细胞耗能大有关,而淋巴管壁耗氧量高于腔静脉管壁是与淋巴管保证淋巴液回流,及淋巴结产生免疫球蛋白等物质与淋巴管内皮细胞直接接触需要消耗能量有关。     

失血性休克对大鼠血管平滑肌BKCa通道酪氨酸磷酸化水平的影响

目的:探讨失血性休克对大鼠肠系膜上动脉血管平滑肌BK_Ca通道酪氨酸磷酸化的影响以及NO对BK_Ca通道酪氨酸磷酸化的调控。方法:建立大鼠失血性休克模型[(35±5)mmHg]并提取肠系膜上动脉总裂解蛋白,采用免疫沉淀及免疫印迹技术,观察休克血管平滑肌BK_Ca通道酪氨酸磷酸化的变化情况;采用原代培养的大鼠肠系膜上动脉血管平滑肌细胞,观察NO对BK_Ca通道酪氨酸磷酸化的调控及其时-效、量-效关系。结果:失血性休克2 h及4 h后大鼠肠系膜上动脉血管平滑肌BK_Ca通道α亚基酪氨酸磷酸化明显增强 (P＜0.01 );L-精氨酸(5×10^-5-5×10^-4 mol/L)孵育30 min即可诱导培养血管平滑肌细胞BK_Ca通道α亚基酪氨酸磷酸化增强,2 h内无明显下降;且L-精氨酸诱导BK_Ca通道α亚基酪氨酸磷酸化具有剂量依赖性。结论:重症失血性休克可增强血管平滑肌BK_Ca 通道酪氨酸磷酸化,且NO参与了该调控过程。     

小鼠肺微血管内皮细胞磁珠分选法分离和原代培养

背景:肺组织的功能依赖于肺微血管内皮细胞的活性,因此肺微血管内皮细胞是相关研究的重要细胞模型,但目前国内多采用的组织块贴壁法培养所得的肺微血管内皮细胞常有其他细胞混杂。目的:建立一种有效分离、培养、扩增小鼠肺微血管内皮细胞的方法。方法:采用酶消化、免疫磁珠二次分选法分离纯化小鼠肺微血管内皮细胞,贴壁培养法体外扩增,CCK-8法测定细胞的生长情况,相差显微镜观察培养细胞的形态,透射电子显微镜观察细胞的超微结构,流式细胞仪对其表型进行鉴定。结果与结论:培养所得小鼠肺微血管内皮细胞具有典型的铺路石样形态学特征,含有大量内皮细胞特有的杆状细胞器Weibel-Palade小体,较稳定地表达内皮细胞特异性表面标记CD105,不表达淋巴管内皮细胞特异性表面标记血管内皮生长因子受体3。说明免疫磁珠二次分选法可成功分离纯化小鼠肺微血管内皮细胞,体外培养所获细胞纯度高、自我更新能力强,并保留了包括构成、表面抗原表达等特性。     

脱细胞血管组织基质的制备和平滑肌细胞的种植

目的组织工程血管的研制成功将为先天性心脏病外科提供一种新的理想的修补材料,本文研究血管脱细胞组织基质的制备,平滑肌细胞的体外培养和种植方法.方法取成年羊胸主动脉,用胰酶消化去除细胞成分,保存弹性蛋白和胶原蛋白,获得血管脱细胞组织基质,经点状注射法,种植体外培养的小牛平滑肌细胞.结果羊主动脉经脱细胞处理,得到的组织基质最大负载下降20%,最大伸长无显著改变;脱细胞组织基质的胶原蛋白含量与新鲜主动脉相似;基质的纤维结构完整,为网状或多孔状;种植的小牛平滑肌细胞生长良好.结论采用多步骤方法获得的血管脱细胞组织基质可用作为制备组织工程血管的支架,适宜于血管平滑肌细胞的生长.     

人血管平滑肌细胞种植构建工程生物血管的实验研究

了解猪血管去细胞后平滑肌细胞种植情况,为猪血管用于血管组织工程提供资料。取猪颈动脉,生物酶预处理猪血管,在自行设计制作的新型动力性生物反应器中,用原代培养的人平滑肌细胞种植在去细胞血管基质材料内,HE染色及银染检测平滑肌细胞种植效果。结果表明生物酶预处理血管后,HE染色及银染检测可见血管腔平滑肌细胞形态正常,沿血管长轴分布,提示经生物酶预处理的猪血管人平滑肌细胞能成功种植,可望构建实用的组织工程血管。     

香烟提取物影响大鼠主动脉平滑肌细胞结缔组织生长因子的表达

背景：血管平滑肌细胞增殖是心血管系统组织变化的基础。 目的：观察不同浓度香烟提取物对大鼠主动脉平滑肌细胞结缔组织生长因子表达的影响。 方法：取第3代大鼠主动脉平滑肌细胞,分别用体积分数2.5%,5%,10%,20%的香烟提取物进行干预。 结果与结论：实时定量RT-PCR结果显示,体积分数2.5%,5%,10%,20%的香烟提取物作用12 h均可促进大鼠主动脉平滑肌细胞结缔组织生长因子mRNA的表达,以体积分数5%香烟提取物的作用最明显,且其刺激大鼠主动脉平滑肌细胞结缔组织生长因子mRNA的表达在24 h内呈时间依赖性。提示香烟提取物可上调大鼠主动脉平滑肌细胞结缔组织生长因子mRNA的表达,导致大鼠主动脉平滑肌细胞的增殖。     

尿源性干细胞在体外向尿路上皮细胞和平滑肌细胞的分化

文题释义：干细胞分化：干细胞通过体外诱导的方法,基因组在时间和空间上选择性表达,产生特异性蛋白质,定向成为特定的成熟细胞。 平滑肌：是由平滑肌细胞组成的非横纹肌肌肉组织,分布在动脉和静脉血管管壁、膀胱、子宫、男性和女性生殖道、消化道、呼吸道、眼睛的睫状肌和虹膜。 背景：成体干细胞是存在于已分化组织中的多能干细胞,尿源性干细胞为新发现的成体干细胞,具有取材方便、高度增殖能力、多向分化潜能等优点,在组织工程学领域受到重视。 目的：研究人尿源性干细胞体外分离获取的方法,以及体外向尿路上皮细胞和平滑肌细胞分化的可行性。 方法：收集健康成人尿液标本,通过分离、培养获取尿源性干细胞;应用CCK-8实验检测细胞增殖能力,并绘制细胞生长曲线;应用流式细胞仪检测细胞表型;体外应用专用培养基诱导尿源性干细胞向尿路上皮细胞和平滑肌细胞分化;应用荧光定量PCR、免疫组织化学染色、免疫荧光细胞染色和Western blot鉴定尿源性干细胞的分化能力。 结果与结论：①从健康成人尿液中成功分离提取出尿源性干细胞,细胞具有高度增殖能力,生长曲线呈现S型;②尿源性干细胞高表达间充质干细胞表面标记物,CD29表达率为98.11%,CD90表达率为95.74%;③尿源性干细胞经体外诱导分化14 d后,可以表达尿路上皮细胞特异性基因Cytokeratin 7、Cytokeratin 20、Uroplakin Ⅲ和平滑肌细胞特异性基因α-SMA和SM22;④结果表明,尿源性干细胞经过体外诱导可以向尿路上皮细胞和平滑肌细胞分化,能够为尿路组织修复重建提供理想的种子细胞来源。 ORCID: 0000-0002-6460-3332(赵战魁) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

平滑肌细胞在旋转生物反应器内的微载体培养与快速扩增

为获取足量数量活性良好的膀胱平滑肌种子细胞，本实验探索在旋转生物反应器内应用微载体技术快速扩增膀胱平滑肌细胞的方法。将培养的第二代新西兰兔膀胱不滑肌细胞应用Cytodex-3微载体在旋转生物反应器（RCCS）内进行动态培养，观测平滑肌细胞的生长情况和细胞代谢率，进行a-肌动蛋白免疫组化染色，并与常规方法培养平滑肌细胞进行比较。结果显示膀胱平滑肌细胞在Cytodex-3微载体上生长迅速、细胞代谢率高、生长倍增时间缩短。在培养第九天，细胞数量可达最初接种的23倍。免疫组化显示平滑肌细胞活性良好。结果表明利用微载体细胞培养技术可简便快速地在体外扩增平滑肌细胞，可为构建组织工程化人造膀胱提供大量活性良好的平滑肌细胞。     

Morphologic characterization of cultured smooth muscle cells isolated from the tracheas of adult dogs

The goals of our study were to isolate smooth muscle cells from the trachealis muscle of adult dogs and to characterize the cells morphologically when they were maintained in primary culture. Enzymatic digestion of the muscle yielded 4.8 +/- 1.8 X 10(6) viable smooth muscle cells per gram of tissue. When placed in culture, these cells rapidly proliferated until confluence was reached. The proliferating cells in culture differed from the cells in the intact tissue in that they stained less intensely for smooth muscle myosin, developed immunofluorescent staining for the intermediate filament protein vimentin, and lost many of the ultrastructural properties of the intact muscle. Only within nodules of cells in the confluent cultures were these ultrastructural properties preserved. Cultures of canine tracheal fibroblasts differed from these smooth muscle cell cultures in that the fibroblasts did not stain for smooth muscle myosin and did not form nodules at confluence. We concluded that adult canine airway smooth muscle cells may be maintained in primary culture, that the confluent cultures contain nodules of cells with many morphologic characteristics of the intact muscle, and that these preparations may be distinguished from cultured canine tracheal fibroblasts on specific morphologic grounds.     

Engineering of vaginal tissue in vivo

Congenital vaginal anomalies and cloacal malformations may require extensive surgical reconstruction. Surgical challenges are often encountered because of the limited amounts of native tissue available. We investigated the feasibility of using vaginal epithelial and smooth muscle cells for the engineering of vaginal tissues in vivo. Vaginal epithelial and smooth muscle cells of female rabbits were grown, expanded in culture, and characterized immunocytochemically. Vaginal epithelial and smooth muscle cells were seeded on polyglycolic acid (PGA) scaffolds at 10 x 10(6) and 20 x 10(6) cells/cm(3), respectively. The cell-seeded scaffolds were subcutaneously implanted into nude mice. The animals were killed 1, 4, and 6 weeks after implantation. Immunocytochemical and histochemical analyses were performed with pancytokeratins AE1/AE3 and with smooth muscle-specific alpha-actin antibodies to confirm the reconstituted tissue phenotype. Western blot analyses and electrical field stimulation studies were also performed to further characterize the tissue-engineered constructs. Vaginal epithelial cells were serially identified with anti-pancytokeratins AE1/AE3 at all culture stages. Smooth muscle cells in culture stained positively with alpha-smooth muscle actin antibodies. One week after implantation in vivo, the retrieved polymer scaffolds demonstrated multilayered tissue strips of both cell types, and penetrating native vasculature was also noted. Increased organization of the smooth muscle and epithelial tissue was evident by 4 weeks. There was no evidence of tissue formation in the controls. Immunocytochemical analyses using anti-pancytokeratins confirmed the presence of vaginal epithelial cells in each of the constructs. Anti-alpha-actin smooth muscle antibodies also confirmed the presence of multilayered smooth muscle fibers and tissue at each time point. Western blot analyses of the scaffolds confirmed the expression of cytokeratin and smooth muscle actin proteins when compared with controls. The contractile properties of the tissue-engineered vaginal constructs in response to electrical field stimulation were similar to those of normal vaginal tissue. Vaginal epithelial and smooth muscle cells can be easily cultured and expanded in vitro. Cell-seeded polymer scaffolds are able to form vascularized vaginal tissue in vivo that have phenotypic and functional properties similar to those of normal vaginal tissues. This is the first demonstration in tissue engineering wherein vaginal epithelial and smooth muscle cells are reconstituted in vivo into vaginal tissue. This technology may be pursued further experimentally in order to achieve the engineering of vaginal tissues for clinical applications.     

Perfusion bioreactor for small diameter tissue-engineered arteries

A scaleable perfusion bioreactor has been developed for tissue engineering of small diameter arterial constructs. This modular bioreactor allows for dynamic sequential seeding of smooth muscle and endothelial cells, biomechanical stimulation of cells during culture, and monitoring of tissue growth and maturation. Bovine aortic smooth muscle and endothelial cells were seeded onto porous tubular poly(glycolic acid) nonwoven scaffolds and cultured in the bioreactor under pulsatile flow conditions for up to 25 days. Cell proliferation was more than 3-fold after 4 days, smooth muscle cells expressed differentiated phenotype after 16 days, and collagen and elastin were distributed throughout the construct after 25 days of culture. In bioreactor experiments in which the construct lumen was seeded with endothelial cells by perfusion after 13 days of smooth muscle cell culture, endothelial cell seeding efficiency was 100%, and a confluent monolayer was observed in the lumen within 48 h. These data demonstrate that this perfusion bioreactor supports sequential seeding of constructs with smooth muscle and endothelial cells. Dynamic culture under pulsatile flow leads to cellular expression of differentiated function and extracellular matrix deposition toward the development of tissue-engineered arterial constructs.     

Development of an intestinal cell culture model to obtain smooth muscle cells and myenteric neurones

This paper reports on the development of an entirely new intestinal smooth muscle cell (ISMC) culture model using rat neonates for use in pharmacological research applications. Segments of the duodenum, jejunum and ileum were obtained from Sprague-Dawley rat neonates. The cell extraction technique consisted of ligating both ends of the intestine and incubating (37 degrees C) in 0.25% trypsin for periods of 30-90 min. Isolated cells were suspended in DMEM-HEPES, plated and allowed to proliferate for 7 days. Cell culture quality was assessed via a series of viability tests using the dye exclusion assay. In separate experiments, tissues were exposed to trypsin for varying durations and subsequently histological procedures were applied. Cell purification techniques included differential adhesion technique for minimizing fibroblasts. Selective treatments with neurotoxin scorpion venom (30 microg mL(-1)) and anti-mitotic cytosine arabinoside (6 microm) were also applied to purify respectively ISMC and myenteric neurones selectively. The different cell populations were identified in regard to morphology and growth characteristics via immunocytochemistry using antibodies to smooth muscle alpha-actin, alpha-actinin and serotonin-5HT3 receptors. Based on both viability and cell confluence experiments, results demonstrated that intestinal cells were best obtained from segments of the ileum dissociated in trypsin for 30 min. This provided the optimum parameters to yield highly viable cells and confluent cultures. The finding was further supported by histological studies demonstrating that an optimum incubation time of 30 min is required to isolate viable cells from the muscularis externae layer. When cell cultures were treated with cytosine arabinoside, the non-neuronal cells were abolished, resulting in the proliferation of cell bodies and extended neurites. Conversely, cultures treated with scorpion venom resulted in complete abolition of neurones and proliferation of increasing numbers of ISMC, which were spindle-shaped and uniform throughout the culture. When characterized by immunocytochemistry, neurones were stained with antibody to 5HT3 receptors but not with antibodies to alpha-smooth muscle actin and alpha-actinin. Conversely, ISMC were stained with antibodies to alpha-smooth muscle actin and alpha-actinin but not with antibody to 5HT3 receptors. The present study provides evidence that our method of dissociation and selectively purifying different cell populations will allow for pharmacological investigation of each cell type on different or defined mixtures of different cell types.