尿道粘膜上皮细胞的分离培养和鉴定

目的：为采用组织工程技术构建尿道粘膜组织提供实验依据。方法：取雄性新西兰幼兔尿道粘膜组织小块,酶消化成单细胞悬液,接种后静置培养、传代。动态观察细胞形态变化及生长增殖情况。细胞进行常规组化染色、免疫组化染色及流式细胞仪检查,并观察超微结构。结果：整个上皮细胞生长期内无成纤维细胞混杂生长,均为单一的上皮细胞,并证实为二倍体细胞。细胞可传11－13代,成活50－60d。结论：新西兰幼兔尿道粘膜上皮细胞可在体外培养,在一定时间内保持增殖能力。     

人输卵管上皮细胞体外无血清培养

输卵管是早期胚胎发育的重要场所，在研究人类生殖生理等方面有重要的作用。本实验取要求行绝育术或子宫肌瘤行子宫全切病人的输卵管组织，用胶原酶消化分离输卵管上皮细胞，在有血清培养液中进行原代培养获得成功，并利用无血清培养液培养传代细胞，建立了人输卵管上皮细胞体外无血清培养的模型。无血清体外培养细胞传代５次，最长生存时间达４０天。     

膀胱黏膜上皮细胞体外培养的实验研究

目的探索组织工程化尿道黏膜类似组织体外培养条件.方法取雄性大耳白兔幼兔的膀胱壁组织,分别用Ⅱ型胶原酶和Dispase Ⅱ处理后,胰蛋白酶消化获得单细胞悬液,接种后用含有或不含有表皮生长因子的培养液培养并传代,动态观察细胞形态变化及生长增殖的不同情况,并对细胞进行超微结构观察.结果用Ⅱ型胶原酶处理的标本在有表皮生长因子存在的情况下,细胞生长良好,混杂少量的成纤维细胞,在体外可传10代以上,成活30天以上.结论Ⅱ型胶原酶处理的幼兔膀胱黏膜上皮细胞纯度较高,可在体外培养一定时间并保持增殖能力,表皮生长因子是必要条件.     

人表皮干细胞的体外分离培养和鉴定

目的:探讨人表皮干细胞的体外快速分离培养及鉴定方法。方法:中性蛋白酶和胰蛋白酶两步法从手术切除的人包皮组织中分离表皮层和真皮层,并获得表皮单细胞悬液,采用Ⅳ型胶原铺板选择性粘附、分离和角质形成细胞无血清培养基(K-SFM)培养表皮干细胞。倒置显微镜下观察培养细胞的生长状况,检测细胞克隆形成率,免疫组化染色观察表皮干细胞标志物β1整合素和角蛋白19(K19)的表达;以角质形成细胞作为对照。结果:组织学观察显示,培养24h后细胞呈克隆状生长;所分离、培养细胞的克隆形成率高于对照角质形成细胞组;免疫组化染色显示,培养细胞β1整合素及Kl9均呈阳性表达。结论:运用Ⅳ型胶原粘附结合K-SFM培养可以实现人表皮干细胞的体外快速分离和培养。     

阴道粘膜上皮细胞的分离培养和鉴定

目的 初步建立阴道粘膜上皮细胞体外培养方法,为阴道粘膜上皮研究提供实验模型.方法 取雌性新西兰大白兔阴道粘膜组织小块,胶原酶Ⅳ和胰蛋白酶联合消化分离法收集上皮细胞,接种于角朊细胞无血清培养液中静置培养、传代.动态观察细胞生长增殖情况,扫描和透射电镜观察超微结构,流式细胞仪测定细胞增殖周期,并进行免疫组织化学鉴定.结果 体外培养的阴道粘膜上皮细胞为二倍体细胞,增殖状态良好,细胞间可见桥粒连接,免疫组化角蛋白染色阳性.细胞的超微结构和免疫组化染色均具有上皮细胞特征.结论 在本实验条件下,体外培养的阴道粘膜上皮细胞具有较好的增殖能力,可作为阴道粘膜上皮研究的理想实验模型.     

膀胱移行上皮细胞的体外培养研究

目的：探索并建立大鼠膀胱移行上皮细胞的体外培养方法，为膀胱癌相关研究提供实验依据。方法：获取Wistar大鼠膀胱粘膜，采用Dispase酶消化法收集上皮细胞。动态观察细胞形态变化、生长增殖情况；对培养细胞进行细胞周期检测及超微结构观察，并行免疫组织化学鉴定。结果：培养细胞为二倍体移行上皮细胞，无成纤维细胞混杂生长。免疫组织化学证实角蛋白染色阳性。结论：膀胱移行上皮细胞体外培养为体外研究提供了理想的模型。     

猪气管上皮细胞的分离、培养和鉴定

目的 探索猪气管上皮细胞体外适宜的生长、增殖条件,以期为组织工程化气管内壁上皮化提供种子细胞.方法 无菌切取猪颈段气管,细菌蛋白酶消化.以明胶作为铺层,接种、培养.待细胞贴壁后,进行光镜观察、广谱角蛋白免疫组化检测、免疫荧光鉴定.结果 光镜观察可见细胞呈铺路石样生长.角蛋白检测和免疫荧光检测均呈阳性,而阴性对照组和空白对照组呈阴性.结论 以明胶作为铺层可以培养出活力较好、纯度高的气管上皮细胞.     

人类肥大乳房乳腺上皮细胞的原代培养

目的 探讨人类肥大乳房乳腺上皮细胞的原代培养方法.方法 采用胶原酶消化培养法,在体外进行人类肥大乳房乳腺上皮细胞的分离培养,用倒置显微镜观察、细胞爬片、HE染色和细胞角蛋白免疫组织化学染色的方法对分离培养的细胞进行形态学观察和鉴定.结果 倒置显微镜下观察细胞形态呈鹅卵石型或多角型,部分形态不规则,增殖的过程中形成岛屿状闭合型的细胞群,细胞之间连接紧密.细胞HE染色可见细胞胞质呈粉红色或浅紫色,细胞核呈蓝紫色,圆形或椭圆形,核中可见深蓝色的染色体.免疫组化鉴定结果显示,培养的细胞胞浆内可见棕黄色的细胞角蛋白着色,表达上皮细胞特异的细胞角蛋白18.结论 应用酶消化法和条件培养液可以获得纯度较高的人类肥大乳房乳腺上皮细胞.     

大鼠肺泡Ⅱ型上皮细胞的分离、纯化和鉴定

目的：探讨大鼠肺泡Ⅱ型上皮细胞（ATⅡ）分离、培养及鉴定的方法。方法：采用Dobbs法提取ATⅡ。肺动脉灌洗减少肺内红细胞,气管灌洗除去肺泡腔内的白细胞,将胰蛋白酶、胶原酶灌入肺内消化分离细胞;采用免疫贴附法纯化细胞,将细胞悬液培养于覆被IgG的平皿中,有Fc段受体的细胞被黏附,而无Fc段受体的ATⅡ得以纯化。ATⅡ的鉴定采用电镜和肺表面活性物质相关蛋白A（SP-A）免疫组化染色,其在电镜下有特征性板层小体,免疫组化染色见胞浆有SP-A表达。结果：纯化后台盼蓝染色显示细胞活力为95%以上。通过SP-A免疫组化染色判定,纯化后ATⅡ纯度可达92%。结论：分离、纯化大鼠ATⅡ时,合适的胰蛋白酶浓度及作用时间对细胞活性有重要作用,大鼠IgG黏附纯化可以得到高纯度的ATⅡ,通过电子镜观察板层小体和免疫组化染色检测细胞SP-A表达可用于鉴定ATⅡ。     

改良人输卵管上皮细胞培养法及电镜观察

目的简化人输卵管上皮细胞分离及培养方法,提高上皮细胞纯度。方法收集19例子宫肌瘤或子宫腺肌病等手术切除的输卵管进行上皮细胞体外培养。改良酶消化法和刮取法收集人输卵管上皮细胞,采用溶血法及差速贴壁法纯化上皮细胞。观察上皮细胞生长及特征,免疫组织化学检测其纯度,扫描和透射电镜评价细胞超微结构。结果改良法培养的人输卵管上皮细胞占细胞总数>90%;电镜显示原代培养的细胞具有微绒毛及纤毛等上皮细胞特征,部分纤毛细胞的胞膜游离面有球状突起,细胞内可见含电子致密物或暗物质的囊泡。结论本改良法方法简单,培养的人输卵管上皮细胞纯度高,电镜结果有利于输卵管上皮细胞分化的研究。     

人骨髓来源内皮祖细胞的分离培养及生物学特性的鉴定

目的建立人骨髓来源的内皮祖细胞（EPCs）分离、培养、诱导分化与鉴定的方法,并探讨其生物学特性。方法收集腰椎间盘退变性疾病患者的髂骨骨髓24例,密度梯度离心法分离的单个核细胞接种于纤维连接蛋白包被的培养瓶,贴壁培养,EGM-2培养基诱导扩增EPCs。Dil-ac-LDL、FITC．UEA—I荧光双标、流式细胞术鉴定EPCs,计算纯度。透射电镜和管腔形成实验鉴定EPCs向血管内皮细胞（ECs）分化的能力。结果培养72h换液时可见贴壁细胞形成明显细胞克隆集落,第5天集落数增加,1周后细胞融合达80％,至第14天细胞呈铺路石样排列。培养至第7天细胞Dil-ac-LDL、FITC-UEA-I双荧光染色阳性率为（95．1±4．0）％,CD133、CD34、KDR、VE。Cadherin标记阳性率分别为（18．5±4．4）％、（45．4±7．8）％、（66．7±7．2）％、（20．5±5-3）％。培养第10天细胞透射电镜显示成熟血管ECs特有的Weibel-Palade小体。管腔形成实验表明,体外ECMatrix上培养7至12d的EPCs具有较强的管腔形成能力。结论采用密度梯度离心与贴壁培养法分离、培养的人髂骨骨髓来源的单个核细胞在特定诱导条件下可分化为EPCs,纯度较高,稳定性和重复性好。EPCs培养7至12d,具有良好的管腔形成能力。     

表皮干细胞的分离培养和鉴定

目的分离培养和鉴定人表皮基底层干细胞。方法中性蛋白水解酶选择性的消化表皮与真皮之间的细胞连接,采用改良的Ⅳ型胶原铺板选择黏附法分离、培养表皮中的干细胞,观察培养第2、4、6天时a6、β1整合素、K19、K14、p63、Nestin、CD34、PCNA及K10在表皮干细胞中的表达差异。结果改良过的Ⅳ型胶原铺板选择黏附法能够有效地促进表皮干细胞的贴壁和伸展。原代分离的表皮基底层干细胞a6、β1整合素,K19,K14,p63,Nestin,CD34及PCNA表达为强阳性而K10不表达。这些细胞具有干细胞的特点,可以形成克隆。随着培养时间的增加,表皮干细胞形态发生变化,a6、β1整合素、K19、K14,表达逐渐减弱,K10表达逐渐增强。此外,在原代分离的细胞中可见单个核样干细胞表达a6、β1整合素,K19,K14,p63,Nestin,CD34及PCNA,这些细胞形态相似体积较表皮基底层干细胞大,胞核为肾形,染色较深。结论中性蛋白水解酶消化合并改良过的Ⅳ型胶原铺板选择黏附法能够高效地分离表皮中的干细胞。分离的细胞具有干细胞的特点,可以形成集落。此外,原代分离培养的表皮干细胞中仍然可见单个核样干细胞,这些细胞形态相似,类似血液系统来源的单核细胞,胞质内均匀分布着粗大的阳性颗粒。单个核样干细胞可能与皮肤的发生、发育以及创伤修复有关。     

Comparison of fresh and cryopreserved human sperm attachment to bovine oviduct (uterine tube) epithelial cells in vitro.

Formation of a prefertilization sperm reservoir in mammals is thought to occur via sperm cell attachment to fallopian tube or oviduct epithelial cells (OEC). Recent data suggests that such an interaction also occurs for human sperm in the fallopian tube. We have previously validated an in vitro sperm-OEC coculture system utilizing bovine OEC monolayers to study postejaculatory human sperm physiology. This study was done to evaluate aspects of human sperm attachment to OEC in coculture and to determine if such attachment and subsequent sperm survival differ between fresh and cryopreserved human sperm. In experiment 1, aliquots of fresh (n = 4) or cryopreserved sperm (n = 3) from normospermic donors were placed into coculture with OEC monolayers at dilutions ranging from 2 x 10(5) to 15 x 10(6) sperm per well. Numbers of each type of sperm attaching to OEC at each concentration were determined. In experiment 2, fresh and cryopreserved sperm from the same donors (n = 4) were put into OEC coculture to observe numbers attaching and subsequent survival time for each sperm type. Sperm attachment to OEC occurred in a linear, dose-dependent manner for fresh and cryopreserved sperm in experiment 1, both as a function of total sperm numbers and as a function of numbers of motile sperm applied (R2 > or = 0.79). However, cryopreserved sperm attached to the OEC at a slower rate than fresh (as a function of the average increase in the number of sperm attaching per unit increase in the number of sperm applied; P < 0.05), with an overall lower percentage of the total and motile sperm applied attaching to OEC (P < 0.01) for cryopreserved versus fresh sperm. Fewer cryopreserved sperm also attached to the OEC, as compared with fresh sperm, in experiment 2 (P < 0.05), even after correcting for motility differences between the sperm types. Sperm survival time in coculture was also decreased for cryopreserved sperm as compared with fresh sperm (P = 0.005). Understanding the kinetics of sperm and OEC interactions may be useful for developing improved cryopreservation protocols or bioassays of sperm function.     

Neuroendocrine differentiation of human prostatic primary epithelial cells in vitro

BACKGROUND: Dispersed prostatic neuroendocrine cells are involved in growth regulation of the prostate and are considered to play a role in the pathogenesis of prostate carcinoma and benign prostatic hyperplasia (BPH). They are meant either to be derived from the neural crest during embryogenesis or by direct differentiation of the cells from locally present precursor cells. METHODS: An in vitro model was developed for human prostatic epithelial and neuroendocrine cell differentiation. Minced explants from radical prostatectomies were seeded on collagen I-coated plates. RESULTS: The majority of outgrowing cells were basal cells, positive for cytokeratin markers K 5/14 and CD 44, as determined by confocal laser scanning microscopy. A small fraction of interdispersed single cells expressing c-kit, which is found on pluripotent precursors, was identified by immunofluorescence. From these basal cells, in vitro differentiation of cells with neuroendocrine morphology could be achieved within 3 days. These were at rest, i.e., non-bromodeoxyuridine incorporating cells and characteristically coexpressed K 5/14, K 18, and the neuroendocrine marker chromogranin A. Luminal cells staining for K 8 or 18 were not observed. CONCLUSION: Neuroendocrine differentiation of adult prostatic cells was achieved in vitro, favoring the hypothesis that neuroendocrine cells are derived from peripheral precursor cells. The acceleration of this differentiation pathway may be the reason for the increased presence of neuroendocrine cells in areas of epithelial hyperplasia in BPH.     

微囊化人头皮毛乳头细胞体外培养及异种移植

目的探讨微囊化人头皮毛乳头细胞体外培养及异种移植的可行性,并对海藻酸钠-多聚赖氨酸-海藻酸钠（alginate-polylysine-alginate,APA）微囊与海藻酸钠-BaCi2（barium-alginate,BA）微囊的物理、生物性能进行评价.方法采用“一步酶消化法”分离、培养人头皮毛乳头细胞,分别用APA微囊与BA微囊包裹.对两种微囊的生物相容性、机械强度、免疫隔离效果及微囊内细胞活性进行比较.结果APA微囊生物相容性优于BA微囊（P＜0.01）,但机械强度低于BA微囊（P＜0.01）;成囊后短期BA微囊内细胞活性高于APA微囊（P＜0.01）,但APA微囊内细胞活性增高较快（P＜0.05）.在微囊完整、表面无纤维化时,两种微囊均可起到良好的免疫隔离作用.结论微囊化人头皮毛乳头细胞可在体外及异种体内培养.综合评价APA微囊与BA微囊的利弊,在不同情况下选择不同的成囊方式是必要的.     

In vitro formation and expansion of cysts derived from human renal cortex epithelial cells.

Acquired renal cysts derive from terminally differentiated tubular epithelium in adults as a consequence of increased epithelial cell proliferation, fluid accumulation and extracellular matrix remodelling. To understand better how human epithelial cysts may be initiated and progressively expand, cells from primary cultures of normal human adult renal cortex were dispersed in polymerized type I collagen. The transparent matrix permitted repeated observation by light microscopy of cyst formation from individual renal cells. The cyst cells reacted strongly with distal nephron histochemical markers (cytokeratin antibodies AE1/AE3, epithelial membrane antigen, and Arachis hypogaea lectin) but inconsistently or not at all to markers of proximal tubules (Tetragonolobus purpureas lectin and Phaseolus vulgaris erthroagglutinin lectin). The number of spherical, fluid-filled epithelial cysts that developed in a standardized microscope field quantified cyst initiation. Cyst progression was determined from the increase in the diameter (surface area) of cysts and represents a hyperplastic event. EGF or TGF alpha, were required in serum-free defined medium to cause cysts to develop from individual epithelial cells dispersed in the matrix; insulin was required as a co-factor. The EC50 for EGF was approximately 0.1 ng/ml, and for insulin 1 microgram/ml. Early cultures of normal cortex formed cysts more efficiently when dispersed in collagen matrix than cells passaged several times before suspension in the gel. Agonists of adenylate cyclase (PGE1, AVP, VIP, PTH, forskolin, cholera toxin), methylisobutylxanthine, and 8-Br-cAMP, though incapable of causing cyst formation alone in defined medium, enhanced cyst initiation and progression in the presence of EGF and insulin. Angiotensin II, TNF alpha, beta-estradiol, and pertussis toxin had no effect in the absence or presence of EGF and insulin. Pertussis toxin inhibited cyst initiation and expansion caused by EGF and forskolin but potentiated cyst initiation and expansion caused by EGF and PGE1. Cyst formation and expansion were inhibited by TGF beta 1 and 2-chloroadenosine. Polarized monolayers of human renal cortical cells grown on permeable membranes were used to independently quantify the effects of agonists on the net secretion of solute and water from the basolateral to the apical surface of the cells. PGE1, forskolin, and 8-Br-cAMP stimulated net fluid secretion that was sustained for several days; EGF enhanced forskolin-stimulated fluid secretion. We conclude that the formation and expansion of in vitro cysts derived from solitary human cortex cells depends on the coordinated interplay between cellular proliferation and fluid secretion.(ABSTRACT TRUNCATED AT 400 WORDS)     

人脐血中血管内皮祖细胞体外扩增和鉴定的实验研究

目的：探讨从人脐血中分离、培养、体外扩增血管内皮祖细胞(EPC)的方法及体外进行EPC的鉴定。方法：采用密度梯度离心法从脐血中分离EPC，体外分别培养在包被有FN和不包被FN的培养皿中，采用免疫组织化学技术(SABC法)鉴定培养贴壁细胞表面标志CD31、CD34及KDR的表达；流式细胞仪检测细胞表面标志CD133及表达CD133细胞比例。结果：包被FN的培养皿中细胞贴壁及增殖均比未包被的多，免疫组化染色CD31、CD34及KDR均呈阳性，培养第4天流式细胞仪检测CD133^ 占21．3％。结论：密度梯度离心法可用于体外分离脐血中EPC进行实验研究，FN对于体外培养EPC有非常重要的作用。     

Pentagastrin stimulates in vitro growth of normal and malignant human colon epithelial cells

Five normal and four malignant human colon epithelial cultures initiated and maintained in our laboratories as well as the standardized in vitro human adenocarcinoma cell line HT-29 were plated in multiwell plates and incubated at 37 degrees C for 72 hours with either phosphate-buffered saline solution or pentagastrin (5 micrograms/ml). Pentagastrin stimulated normal cells to increase (p less than 0.05) in number by an average of 65 percent compared with saline control cells, whereas malignant cells increased an average of 59 percent compared with control cells. There was no difference in the magnitude of trophic effect between the normal and malignant cells. Further studies are indicated to elucidate the role of gastrin in either initiating, promoting, or both, the growth of carcinoma of the colon.     

皮肤源祖细胞的培养、鉴定和体外诱导分化

目的探讨皮肤源祖细胞的体外培养、鉴定及定向诱导成脂、成骨的方法，为组织工程提供较理想的种子细胞。方法出生1～3d的sD大鼠幼鼠皮肤，以含表皮生长因子和成纤维细胞生长因子的培养基进行培养。观察细胞生长情况，描绘生长曲线；免疫荧光鉴定细胞表达Nestin和Fibronectin情况；将第3代细胞，分别用成脂和成骨诱导液培养14d，以油红O染色、茜素红染色和免疫荧光检测皮肤源祖细胞诱导成脂、成骨情况。结果细胞呈悬浮生长，迅速增殖形成克隆球团；细胞免疫荧光表达Nestin和Fibronectin；成脂诱导14d后，细胞内大量致密颗粒形成，油红O染色可见红色脂滴；成骨诱导14d后，茜素红染色可见暗红色钙盐沉积，骨桥蛋白表达阳性显示成骨细胞形成。结论皮肤源祖细胞具有干细胞的特性，能分化为成骨细胞和脂肪细胞。