组织培养下胎儿卵巢雌二醇的分泌

目的 :观察组织培养条件下胎儿卵巢能否分泌雌二醇 (E2 )及胎儿胰腺条件培养液对E2 分泌的影响。方法 :取 16例中晚期妊娠水囊引产的胎儿卵巢 ,将其切成 1 2mm× 1 2mm× 1 2mm的碎块进行直接培养及加胎儿胰腺条件培养液组织培养。培养后测定培养液中E2 含量。结果 :胎儿卵巢在组织培养条件下能产生E2 ,随培养时间延长 ,E2 分泌量增加。加胎儿胰腺条件培养液组 ,E2 分泌量高于对照组 ,P <0 .0 5。结论 :胎儿卵巢在组织培养条件下能分泌E2 ,可作为卵巢移植的供体。     

激活素A和卵泡抑素对冻融人胎儿卵巢雌二醇分泌的影响

目的：研究组织培养条件下冻融人胎儿卵巢雌二醇（E2）的分泌及激活素A（Act A）和卵泡抑素（FS）对E2分泌的影响。方法：冻融人胎儿卵巢组织体外培养，分别加入基础培养液（对照组，即MEM组）、基础培养液＋100ng／ml Act A（Act A组）、基础培养液＋100ng／ml FS（FS组）和基础培养液＋100ng／ml Act A＋100ng／ml FS（A＋F组），每组4块组织，培养8d。收集培养2d、4d、6d、8d各组培养液，测定比较其E2值。结果：培养4d、6d、8d卵巢组织分泌的E2值均高于2d的E2值（均P〈0．05），随培养时间延长，E2分泌量先增加后减少，在培养后6d可达较高分泌水平；Act A组E2分泌量明显高于对照组、FS组和A＋F组（均P〈0．01）。结论：冻融人胎儿卵巢在组织培养条件下能分泌E2，Act A能促进体外培养胎儿卵巢E2的分泌，其作用可能受到FS的调节。     

N乙L-半胱氨酸对胎儿卵巢组织体外培养的影响

目的：探讨硫醇类抗氧化剂 N 乙酰 L 半胱氨酸(n acetyl l cysteine,NAC)对胎儿卵巢组织体外培养效果的影响。方法：取中期引产的20～28周死亡女胎的卵巢,分别在含NAC 25、50、100?mmol/L(实验组)和不含NAC(对照组)的培养液内培养0～9?d,采用液相平衡竞争放射免疫分析法检测各组卵巢组织在不同的培养时间雌二醇（estrogen,E2）分泌量;观察培养后各组卵巢卵泡的形态学变化。结果：培养后卵泡可进一步发育,各期卵泡构成比实验组与未培养组、对照组比较差异均有统计学意义(P<0.05),而3个不同剂量实验组两两之间在卵泡构成比上差异无统计学意义（P>0.05）;胎儿卵巢组织经体外培养后能够分泌E2,E2分泌量实验组和对照组之间差异有统计学意义(P<0.05),随着培养时间的延长E2分泌量显著增加(P<0.05);而在添加不同剂量NAC的3个实验组,两两比较差异则无统计学意义(P>0.05)。结论：在培养液中添加NAC后,E2分泌量增加,各期卵泡的形态得以维持并能进一步发育,为人胎儿卵巢培养后移植提供了实验依据。     

组织培养下胎儿卵巢卵泡的发育

目的 :观察组织培养条件下胎儿卵巢能否发育以及胎儿胰腺条件培养液对其发育的影响。方法 :取 16例 2 0～ 2 8周和 11例 >2 8周中期妊娠水囊引产的胎儿卵巢 ,经直接培养及加胎儿胰腺条件培养液组织培养后 ,观察培养前后的组织学变化。结果 :胎儿卵巢经组织培养后 ,较成熟卵泡比例增加 ,P <0 .0 0 5 ;加胎儿胰腺条件培养液组与未加胎儿胰腺条件培养液组相比 ,较成熟卵泡比例增加更为明显 (P <0 .0 0 5 )。结论 :胎儿卵巢在组织培养条件下能继续发育 ,胎儿胰腺条件培养液有促进卵泡发育的作用。     

兔胚胎卵巢细胞团同种异体移植研究

本实验随机将18只NZW雌兔分为4组。A组(5只):系内移植,供、受体同系,B组(5只):系间移植,供体为NZW封闭群,受体为NZW近交系;C组(5 只);系间移植,供、受体情况同B组,移植物预培养2～3天;D组(3只):移植部位注射等体积培养液,其余条件同前。实验结果显示;①A、B、C3组受体兔在卵巢细胞团移植后,血浆E2水平明显上升(P<0.01),提示移植物有E2分泌;移植后30天内,A组较B、C组E2水平高(P<0.05或<0.01),提示同系移植卵巢功能恢复校好;移植后30～60天,C组E2较B组高(P<0·01),提示培养后移植优于未培养直接移植。②同期移植部位有生长卵泡存在,生殖道组织学也与此结果吻合。     

胎儿卵巢冷冻保存及复苏

目的 :探讨胎儿卵巢的体外保存方法。方法 :将 16例 2 0～ 2 8周的胎儿卵巢置于含 1.41mol/L的二甲基亚砜 (DMSO)培养液中 ,采用三步降温法投入液氮保存 ,复苏时采用 40℃水浴快速复温和室温下慢复温 ,然后进行组织培养并观察卵泡发育和雌二醇 (E2 )分泌情况。结果 :胎儿卵巢冻存后复苏培养 ,其卵泡仍能继续发育并产生E2 ;快速复温组卵泡组织形态良好 ,并能维持良好的内分泌功能 ;慢复温组卵巢上皮破裂 ,E2 分泌量明显减少。结论 :“三步降温法”冻存胎儿卵巢效果好 ,快速复温能更好地维持卵泡形态发育和内分泌功能     

组织培养下胎儿卵巢卵泡的发育

目的：观察组织培养条件下胎儿卵巢能发育以及胎儿胰腺条件培养液对其发育的影响。方法：取１６例２０￣２８周和１１例〉２８周中期妊娠水囊引产的胎儿卵巢,经直接２及加胎儿胰腺条件２液组织培养后,观察培养前后的组织学变化。结果：Ｅ和卵巢经组织２后,罗成熟卵泡比例增加,Ｐ〈０．００５;加胎儿胰腺条件培养液组与未嗷 胰腺条件培养液组相比,较成语卵泡比例增另更为明显。结论：胎儿卵巢在组织培养条件下能继续发育,胎儿     

营养因素对围绝经期卵巢组织雌二醇分泌的影响

目的观察各种营养因素对围绝经期卵巢组织中雌二醇（E2）分泌的影响。方法取围绝经期妇女手术切除的卵巢组织,分别在谷胱甘肽（1mmol/L）、复方氨基酸（550μg/mL）、二者联合应用以及对照组（不含以上营养因素）的无血清培养液中培养9d,检测各组培养液中E2的分泌量。结果培养9d后各处理组E2分泌量与培养3d相比均显著增加（P〈0.05）;培养6d和9d后,谷胱甘肽、复方氨基酸及其联合应用的处理组与对照组相比,E2分泌量显著增加（P〈0.05）。结论谷胱甘肽、复方氨基酸能促进围绝经期卵巢组织分泌E2,并随着作用时间延长效果更明显,从而有可能为围绝经期妇女卵巢保养、女性内分泌疾病诊治提供十分有利的实验依据。     

补肾活血方含药血清对大鼠颗粒细胞类PCOS模型的影响

目的 探讨补肾活血方含药血清对体外培养颗粒细胞类PCOS(多囊卵巢综合症)模型的作用机制. 方法 对大鼠颗粒细胞进行体外分离培养,分成正常组、模型组、含药组、孕马血清组诱导培养,观察细胞形态,收集各组培养液检测E2. 结果 含药组中的类PCOS大鼠颗粒细胞生长良好,E2分泌量最高,与正常组比较差异有显著统计学意义(P<0.01). 结论 补肾活血方含药血清通过促进颗粒细胞的生长增殖和分泌E2的功能,来治疗PCOS.     

人胚甲状旁腺细胞移植供体胎龄的选择

目的研究人胚甲状旁腺细胞移植治疗甲状旁腺功能低下(HPI)中供体最佳胎龄段.方法将12周～32周龄水囊引产的死胎(死亡时间＜2h)的甲状旁腺切取后进行体外培养,定期测量培养液中甲状旁腺激素(PIH)水平,同时对甲状旁腺细胞进行光镜电镜检查,以确定最适合甲状旁腺细胞移植的供体胎龄段.结果16周～24周龄供体的甲状旁腺细胞经体外培养后,其分泌Pm功能良好,其细胞形态及其超微结构保持良好.结论16用～24周龄的供体最适合作甲状旁腺细胞移植.     

组织粘合剂联合羊膜移植在角膜溃疡中的应用研究

目的  观察与探讨氰基丙烯酸组织粘合剂联合羊膜移植在角膜溃疡中的临床疗效。方法  采用前瞻性临床对照研究,将58例接受角膜溃疡羊膜移植术的患者随机分为两组：粘合组33例,用组织粘合剂粘贴羊膜;缝线组25例,用10-0尼龙线缝合羊膜。比较两组手术时间和术后症状,随访6～12个月,观察局部反应、角膜上皮生长情况及羊膜贴附情况等。结果  组织粘合剂组平均手术时间（13.31±2.81）min,比缝线组（18.72±4.33）min明显缩短（P <0.05）。组织粘合剂组术后症状较缝线组轻,羊膜植片较缝线组贴合紧密,其余两组差异无统计学意义。结论  角膜溃疡羊膜移植术中使用氰基丙烯酸组织粘合剂粘合羊膜安全、简便,可代替传统的缝线缝合并在一定程度上优于缝线,值得在临床上进一步研究及推广。

     

Transplantation of human limbal cells cultivated on amniotic membrane for reconstruction of rat corneal epithelium after alkaline burn

Background The transplantation of limbal epithelial cells cultivated on amniotic membrane is a newly developed treatment for limbal stem cell deficiency. The purpose of our study was to investigate the biological characteristics of limbal epithelial cells and evaluate the effect of transplantation of cultivated human limbal epithelial cells on ocular surface reconstruction in limbal stem cell deficiency rat model.Methods Human limbal cells were isolated and cultivated in vitro. Cytokertins 3, 12, and 19 (K3, K12 and K19) and p63 were detected by immunofluorescent staining or RT-PCR. BrdU labelling test was used to identify the slow cycling cells in the cultures. Limbal stem cell deficiency was established in rat cornea by alkali burn.Two weeks after injury, the rats received transplants of human limbal stem cells cultivated on amniotic membrane carrier. The therapeutic effect was evaluated by slit lamp observation, Hemotoxin and Eosin (HE) staining and immunofluorescent staining.Results On day 7 in primary culture, p63 and K19 were strongly expressed by most cells but only a few cells expressed K3. On days 14 and 21, p63 and K19 were still expressed by a majority of cells, but the expressive intensity of p63 decreased in a number of cells, while the proportion of K3 positive cells increased slightly and some cells coexpressed p63 and K3. RT-PCR showed that gene expression of both p63 and K12 were positive in cultivated limbal cells, but in mature superficial epithelial cells, only K12 was detected. BrdU labelling test showed that most cells were labelled with BrdU after 7 days‘ labelling and BrdU label retaining cells were observed after chasing for 21 days with BrdU free medium. For in vivo test, slit lamp observation, HE staining and immunofluorescent staining showed that the rats receiving transplant of human limbal stem cells cultivated on amniotic membrane grew reconstructed corneas with intact epithelium, improved transparency and slight or no neovascularization. A majority of epithelial cells of the reconstructed cornea were positive to antihuman nuclear antibody and cells expressing K3 were found mainly in superfacial epithelium.Conclusions Limbal stem cells can be cultivated in vitro: the cells are characterized by high proliferation and slow cycling and identified as p63/K19 positive and K3/K12 negative. During culture, some stem cells can proliferate and differentiate into mature cornea epithelial cells. Amniotic membrane is a suitable carrier for limbal stem cells. Transplantation of human limbal stem cells cultivated on amniotic membrane can functionally reconstruct rat cornea with limbal stem cell deficiency.     

兔角膜缘上皮细胞培养联合羊膜行自体移植的实验研究

目的：用体外培养的角膜缘上皮细胞联合人羊膜的方法，行自体移植治疗角膜缘干细胞完全缺乏的兔眼。方法：制作10只兔右眼于细胞缺乏的模型，取其中7只兔的左眼上方角膜缘取一小块组织进行体外培养，并传代在无上皮细胞的人羊膜上。1个月后，手术切除10只兔受伤眼角膜表面被覆的新生血管膜和结膜上皮，7眼接受了含自体培养上皮细胞的羊膜移植，另3眼仅移植无上皮细胞的羊膜。结果：1周后原代培养的角膜缘上皮细胞融合，传代至无上皮细胞的人羊膜上继续生长2～3周，角膜上皮细胞可牢固的附着于羊膜上。移植了含有角膜上皮细胞的羊膜的兔子，术后早期都形成了角膜上皮化，并明显抑制了新生血管的再生，而接受羊膜移植的兔子，术后又出现明显的新生血管。结论：利用无上皮细胞的羊膜作为载体，培养角膜缘上皮细胞并自体移植可以恢复角膜上皮化、抑制新生血管生长。     

手术切除联合角膜缘干细胞移植、羊膜移植及单纯手术切除治疗翼状胬肉临床疗效分析

目的 探讨手术切除联合角膜缘干细胞移植、羊膜移植及单纯手术切除治疗翼状胬肉的临床疗效.方法 选择本院2008年3月至2010年5月收治的角膜缘翼状胬肉患者90例,随机分为三组,角膜移植组给予手术切除联合角膜缘干细胞移植,羊膜移植组给予手术切除胬肉联合羊膜移植治疗,单纯手术组仅给予翼状胬肉手术切除；观察三组治疗效果.结果 三组术后随访两年,角膜移植组、单纯手术组术后创面愈合时间短于羊膜移植组,差异有统计学意义(P＜0.05),角膜移植组创面愈合时间与单纯手术组差异无统计学意义(P＞0.05)；角膜移植组术后治愈28例,复发2例,羊膜移植组术后治愈25例,复发5例,单纯手术组术后治愈22例,复发8例,三组术后复发率比较,差异有统计学意义(P＜0.05).结论 手术切除联合角膜缘干细胞移植治疗翼状胬肉,术后复发率低,并发症少,愈合时间短,值得应用.     

不同手术方式治疗翼状胬肉对角膜屈光状态的影响

目的探讨翼状胬肉切除联合自体角膜缘干细胞移植和翼状胬肉切除联合羊膜移植对角膜屈光状态的影响。方法将86例(86眼)翼状胬肉患者随机分为两组,每组43例,A组采用翼状胬肉切除联合自体角膜缘干细胞移植治疗,B组采用切除联合羊膜移植治疗,比较两组患者手术前后裸眼视力、角膜曲率和角膜散光度。结果两组患者术后裸眼视力、角膜曲率较术前提高(P<0.05),角膜散光度较术前减低(P<0.05),三项指标组间比较差异均无统计学意义(P>0.05)。结论翼状胬肉切除联合自体角膜缘干细胞移植和联合羊膜移植均为治疗翼状胬肉的有效的手术方法,临床可根据具体情况选择应用。     

角膜缘干细胞移植与羊膜移植治疗翼状胬肉对比观察

目的:对比观察角膜缘干细胞移植与羊膜移植治疗翼状胬肉的效果。方法:将325例翼状胬肉患者随机分为角膜缘干细胞移植组(A组)和羊膜移植组(B组)。其中A组215例(280眼),B组110例(145眼),术后观察随访6～12个月,统计创面愈合时间及复发情况。结果:A组中有9眼复发,复发率为3.21%;B组有12眼复发,复发率为8.28%。结论:角膜缘干细胞移植与羊膜移植对治疗翼状胬肉均有疗效,其中角膜缘干细胞移植复发率低,角膜创面修复快,效果更好。     

人羊水干细胞移植改善肝硬化大鼠的肝纤维化

目的研究人羊水干细胞（hAFSC）移植对CCl4诱导性肝硬化大鼠的改善。方法使用贴壁法分离培养羊水干细胞,Western blot法鉴定。Wistar大鼠24只随机分为对照组（n=8）和肝硬化组（n=16）,肝硬化组采用60%的CCl4植物油皮下注射7周,制造肝硬化模型,再随机分成肝硬化模型对照组（注射等体积PBS,n=8）、hAFSC移植组（n=8）。直接经肝内注射hAFSC,3周后处死所有大鼠,肝组织石蜡切片进行病理学分析。结果分离的羊水干细胞均表达特异性标记物Oct-4、SSEA-4。与大鼠肝硬化模型组比较。肝硬化程度明显减轻,假小叶附近脂肪化细胞和肝组织中胶原纤维的量明显减少。结论 hAFSC肝内移植可减轻大鼠肝硬化病变程度。     

胎鼠皮肤间充质干细胞的体外培养及成骨分化研究

目的：分离、纯化胎鼠皮肤间充质干细胞(MSCs)，在体外培养、扩增并促其成骨分化，为骨组织工程学研究与骨质疏松症的细胞学治疗提供一种可能。方法：体外培养胎鼠皮肤MSCs，并在培养液中加入成骨诱导剂诱导培养，观察其成骨分化潜能。 结果：胎鼠皮肤MSCs传代培养第15代仍具有增殖能力,说明该细胞为干细胞源性;贴壁细胞在成骨诱导剂作用下,碱性磷酸酶(ALP)水平升高，ALP钙钴染色呈阳性反应，钙结节茜素红染色反应阳性。 结论：胎鼠皮肤MSCs具有很强的自我增殖能力，成骨诱导剂可促其向成骨分化。     

Characterization and safety assessment of bioengineered limbal epithelium

Transplantation of cultivated limbal epithelium on substrates such as amniotic membrane is an established treatment for severe ocular surface disease with limbal stem cell deficiency. In this study, we adapted an established method to generate sheets of limbal epithelium on amniotic membrane and characterized the cells contained in these sheets and tested them for safety with regard to microbial contamination. Human limbal biopsies were cultivated on denuded amniotic membranes. After three weeks of culture, the phenotypes of cultivated cells were analyzed by immunohistochemistry and real-time RT-PCR for the expression of a panel of specific markers. Cultivated limbal epithelial cell sheets were also analyzed by scanning (SEM) and transmission (TEM) electron microscopy. Sterility tests and mycoplasma assays were conducted for the safety of product. A confluent layer of polygonal cells was formed in 2 weeks and 1-3 stratified layer of cells were observed after three weeks of culture. Cultivated cells were positive for p63, K3, K19, and involucrin but negative for K14, integrin alpha9 and ABCG2 when analyzed by immunohistochemistry. Expression of molecular markers was detectable with real-time RT-PCR. SEM showed multilayer of flat squamous polygonal epithelial cells. Desmosomal and hemidesmosomal attachments were evident. Our study showed that cultivated limbal epithelium consists of limbal progenitors as well as differentiated corneal epithelial cells. SEM and TEM analysis showed cultivated cells demonstrated typical features of corneal epithelium. The risk of contamination is low and can be prevented by culturing the cells in a clean room facility complying to Good Manufacturing Practice standard.