大鼠体外多器官细胞共培养模型的建立

目的 建立大鼠体外多器官细胞共培养方法.方法 采用胶原酶消化法、支气管灌洗分离法、两步消化法等方法分离大鼠原代肝细胞、肾细胞、心肌细胞、肺泡巨噬细胞和真皮成纤维细胞,用锥虫蓝染色法检测细胞分离成活率,用单层贴壁法分别培养;采用飞片法,将5种细胞的飞片放入同一培养皿,用体积分数为10%的FBS DMEM培养基培养7 d,用噻唑蓝(MTT)比色法比较原代细胞在单独培养和共培养时的生长情况.结果 建立的大鼠原代肝细胞、肾细胞、心肌细胞、肺泡巨噬细胞、真皮成纤维细胞分离方法稳定,细胞分离成活率均达90%,其中胶原酶消化法培养肝细胞的存活率达90.3%,两步消化法培养肾细胞的细胞存活率达91.9%,心肌细胞的胶原酶消化法细胞存活率达93.0%.3～15 d的心肌细胞搏动频率比较,差异无统计学意义(P>0.05),差速贴壁法培养的肺泡巨噬细胞存活率可达90.8%,以胶原酶消化法培养的原代真皮细胞存活率达92.7%,细胞生长状态良好.5种原代细胞的多器官细胞共培养结果显示,共培养时细胞生长增殖情况良好,单独培养与共培养细胞生长曲线基本重合.结论 所建立的大鼠多器官细胞共培养方法是成功的.     

原代肝细胞模型的优化及苯乙烯和氧化苯乙烯的毒性研究

目的建立具有高活力、高功能特性的原代小鼠肝细胞模型,并通过该体外模型评价受试物苯乙烯和氧化苯乙烯的急性毒性。方法以BABL/C小鼠作为肝细胞供者,在经典两步胶原酶消化法基础上进行优化,通过逆向灌流、间断灌注、限制消化时间及Percoll液离心纯化的方式获取小鼠肝细胞,并进行体外单层和夹层培养;通过细胞形态、细胞活力、胞内糖原颗粒以及上清中各指标即白蛋白(ALB)、乳酸脱氢酶(LDH)、丙氨酸氨基转移酶(ALT)及尿素氮(BUN)变化综合评价原代培养的肝细胞模型;以苯乙烯和氧化苯乙烯为受试物,用浓度分别为0.2、1、5、10和25μmol/L的受试物作用于夹层培养3 d的肝细胞,于染毒3、6、12、24及48 h后,采用CCK-8法和LDH法测定细胞存活率。结果改良法分离获取的小鼠肝细胞活力为(90.3±5.2)%,纯度为(95.3±4.2)%,产量达(2.4±0.9)×107;夹层培养7 d内90%以上细胞呈典型的肝细胞形态特征,培养第3 d胞浆内可见大量糖原颗粒。ALB分泌、LDH和ALT漏出、BUN合成、以及细胞活力夹层培养在8 d内、单层培养在6 d内呈波动性变化,且夹层培养法各指标明显优于单层培养法;夹层培养第3 d ALB分泌量[(1.42±0.20)g/L]和BUN合成量[(1.97±0.22)mmol/L]及细胞活力均达峰值,而LDH漏出量[(7.30±2.33)U/L]和ALT漏出量[(6.51±1.86)U/L]降到谷值,且3~7 d各指标变化相对稳定。苯乙烯和氧化苯乙烯在染毒6 h内,对肝细胞显示了较低的细胞毒性,细胞存活率在90%以上,且CCK-8和LDH两种毒性测定方法之间无显著差异;随着染毒时间的进一步延长,CCK-8法检测出的细胞存活率更低(85%以下),与LDH法相比差异有统计学意义。用不同浓度受试物处理肝细胞24 h后,从5μmol/L开始观察到相对较高的细胞毒性,用CCK-8法检测出细胞存活率约为85%,但与LDH法相比无显著差异;随着染毒浓度的继续增加,CCK-8法检测出的细胞存活率更低(80%以下),与LDH法相比差异有统计学意义。结论改良的胶原酶消化法结合夹层培养法可使肝细胞在较长时间(7 d)内维持良好的形态和功能,且用夹层培养3~7 d的肝细胞模型可以较准确地评价苯乙烯和氧化苯乙烯的毒性效应,结合毒性检测指标推测受试物主要影响肝细胞内线粒体亚细胞器的功能,对肝细胞膜的损伤程度影响较弱。     

20例人早孕绒毛滋养细胞的体外培养

目的建立快速、有效的绒毛滋养细胞体外培养的方法。方法采用0．25％胰酶和Ⅱ型胶原酶混合消化法消化绒毛组织,用胰蛋白酶消化传代体外培养,通过光镜对培养出的细胞进行形态评价;用细胞角蛋白和波形蛋白单克隆抗体检测细胞纯度。结果联合消化法的细胞生长迅速,接种6～8d可以传代,原代细胞生长时间明显缩短,所得细胞纯度达95％。结论联合消化法是一种较有效的人早孕绒毛滋养细胞培养方法。     

肾综合征出血热病毒对人胚肾原代细胞感染作用的研究

近年来对该病进行了广泛研究 ,但其发病机理尚不清楚。为了搞清肾综合征出血热病毒 (ＨＦＲＳＶ)对肾组织的损伤机理 ,本文首次选用易饲养、敏感、高效的人胚肾原代细胞直接攻击ＨＦＲＳＶ。为确保结果的准确性 ,改用了胶原酶温和消化法 ,取得令人满意的结果。现将结果报告如下。1　材料与方法1 1　材料　 (1)ＨＦＲＳ病毒株 :Ｈａｎｔａａｎ病毒 76 / 118株 ,Ｌｅｅ Ｈ .等分离自南朝鲜黑线姬鼠 ,从第四军医大学汪美先教授处引进。昆明种小白鼠乳鼠脑内接种 (0 0 2 /只 )传代 ,取鼠脑研制成 2 0 %病毒悬液。ＬＤ50 =ｌｏｇ6 5 / 0 .0 2ｍ…     

Hep G2培养条件的摸索及其与CHL、3T3细胞代谢能力的比较

目的：以MTT试验方法计算环磷酰胺（cyclophosphamide,CP）对Hep G2细胞和体外安全性评价常用细胞系CHL、3T3细胞的LC50,间接比较几种细胞系的代谢能力并确定Hep G2细胞适宜的培养试验条件.方法：用需代谢活化的阳性物CP以含血清和不含血清的培养液配制染毒液,按10.0、7.21、5.19、3.73、2.69、1.93、1.39、1.00 mg/ml浓度对CHL、3T3细胞和以不同培养液培养的Hep G2细胞染毒,在24 h时间点以MTT方法对细胞存活情况进行分析,计算LC50,比较各种细胞和不同培养条件下Hep G2细胞对CP的代谢活化水平,并观察不同培养条件下Hep G2细胞的生长状态.结果：MTT试验结果表明Hep G2细胞对CP的代谢能力最强,CHL细胞次之,而3T3细胞对CP的代谢能力最弱;3种不同培养液培养的Hep G2细胞无论染毒液是含血清的还是不含血清的均观察到CP对细胞的LC50：MEM＞DMEM＞RPMI 1640,Hep G2细胞在DMEM、RPMI 1640两种培养液中生长状态均良好,尤其是RPMI 1640培养的Hep G2细胞,在无血清的染毒液培养下既能获得较多的细胞又能表现出较强的代谢CP的能力.结论：Hep G2细胞对CP的代谢活化能力较CHL和3T3细胞强,以RPMI 1640培养的Hep G2细胞生长状态良好且较DMEM和MEM培养的细胞表现出更强的代谢活化CP的能力,能获得较理想的培养和试验结果.     

人脐静脉内皮细胞的培养与鉴定

对血管内皮细胞体外培养的常规方法进行了改进 ,采用胰蛋白酶 胶原酶 (1∶1)混合酶液消化方式分离人脐静脉内皮细胞 ,简化完全培养液的组成成分 (不添加血管内皮细胞生长因子、肝素等辅助因子 ) ,机械刮擦法分离单层细胞进行传代培养。培养细胞用形态学、超微形态学和免疫组织化学染色法进行鉴定 ,证实培养的细胞是血管内皮细胞。     

胎鼠骨骼肌卫星细胞的原代培养、纯化与鉴定

目的 探讨胎鼠骨骼肌卫星细胞的原代培养、纯化与鉴定的方法,为胎儿和早产儿骨骼肌发育程序化的研究提供实验基础. 方法 Sprague-Dawley大鼠孕18 d时剖宫取出胎鼠,手术显微镜下取胎鼠四肢骨骼肌肌肉,采用Ⅰ型胶原酶和胰蛋白酶两步消化法分离胎鼠骨骼肌卫星细胞,差速贴壁法进行纯化.原代培养的生长培养基为(DMEM/F12+ 20％ FBS+ 10％ HS+5 ng/ml bFGF).分化培养基为(DMEM/F12+ 2％ HS).CCK-8法绘制胎鼠骨骼肌卫星细胞生长曲线图,细胞免疫组化染色鉴定纯化后细胞的肌源性标志蛋白Desmin,观察分化培养基条件下骨骼肌卫星细胞的成肌分化特性. 结果 胎鼠原代骨骼肌卫星细胞在培养的第3天进入对数生长期,5～6d达增殖平台期;纯化后的细胞90％以上表达结蛋白(Desmin).在分化培养基的诱导下细胞之间相互融合形成具有自发节律性收缩特性的多核肌管. 结论 采用消化法和差速贴壁法分离纯化胎鼠骨骼肌卫星细胞,能够获得高活性和高纯度的具有成肌分化能力的骨骼肌卫星细胞.     

外周血淋巴细胞染色体培养用哪几种培养基为好?

到目前为止,可用作淋巴细胞染色体培养的培养液很多,各实验室的使用情况也略有所异。较常用的有RPMI1640、199、Eagle、F_(10)等,甚至有人用水解乳     

矢车菊-3-O-葡萄糖苷对β淀粉样蛋白25-35致海马神经细胞损伤的干预研究

目的探讨矢车菊-3-O-葡萄糖苷(Cy-3G)的神经保护作用。方法原代培养乳鼠海马神经细胞,免疫荧光法检测细胞纯度。5μmol/Lβ淀粉样蛋白(Aβ)25-35处理细胞3 h,Cy-3G不同方式干预后,噻唑蓝(MTT)法检测细胞存活率,乳酸脱氢酶(LDH)法检测细胞上清液中LDH漏出率,DCFH-DA探针检测细胞内活性氧(ROS)含量,罗丹明123试剂盒检测细胞线粒体膜电位。结果与对照组比,5μmol/L Aβ25-35组细胞存活率显著降低(P0.05),而40μmol/L Cy-3G共孵育3 h可有效提高细胞存活率,还可显著降低细胞上清液中LDH漏出率、升高细胞线粒体膜电位、降低细胞ROS水平(P0.05)。结论 40μmol/L Cy-3G共孵育3 h对Aβ25-35损伤的原代海马神经细胞有保护作用。     

新生小鼠成骨细胞原代培养方法的实验研究

目的 验证及比较成骨细胞原代培养常用方法,探讨两种原代培养方法的可行性和应用价值.方法 取出生后24 h昆明小鼠乳鼠顶骨,以含血清胶原酶消化法和分段胶原酶消化法分离成骨细胞,进行细胞形态观察,以噻唑蓝(MTT)比色实验法绘制生长曲线,以台盼兰排斥法计数活细胞率.结果 与含血清胶原酶消化法相比,分段胶原酶消化法提取的成骨细胞产量多;细胞存活率高(P<0.01);对细胞损伤小,细胞纯度高.消化时问易于控制.结论 分段胶原酶消化法是一种方便、高效、细胞生物学特性稳定的原代成骨细胞分离培养方法.

Abstract：

Objective To confirm an alternative method for primary culture of osteoblasts through comparison of two primary culture methods. Methods Calvarias were dissected from newborn Kunming mice, osteoblasts were isolated with serum-containing collagenase digestion method and sequential digestion method respectively. The osteoblasts were observed under invert microscope, the growth curve was made with the application of MTT method, the rate of living osteoblasts was counted with trypan blue method respectively. Results Compared with the serum-containing collagenase digestion method, the sequential digestion method presented higher production of osteoblasts, higher cell survival rate (P<0.01), little damage on osteoblasts and easier control of the digestion time. Conclusion The sequential digestion of calvaria method is simple, efficient and stable for primary osteoblasts culture.     

大型成年哺乳类动物绵羊阴道平滑肌细胞的原代培养改进方法研究

目的探索原代培养成年大型哺乳动物绵羊阴道平滑肌细胞(vaginal smooth muscle cells,VSMC)改良方法。方法成年绵羊阴道平滑肌取材,利用预消化组织块法(将组织剪碎呈1 mm3大小组织块,0.1%Ⅰ型胶原酶消化20 min,0.1%胰酶-EDTA与0.1%Ⅰ型胶原酶混合溶液再次消化30 min,将组织块种于培养瓶)原代培养VSMC,并与传统组织块法和酶消化法进行细胞萌出速度及增殖速度比较,提高VSMC原代培养效率;免疫荧光染色法和western bloting法检测平滑肌细胞标志物calponin及a-SMA表达鉴定VSMC。结果预消化组织块法获得原代VSMC速度快于传统组织块法和酶消化法,3 d可见细胞萌出,9 d可达80%细胞融合,"峰谷"特征明显,而酶消化法至12 d增殖至80%细胞汇合,传统组织块法约7 d可见细胞萌出,14 d可达80%细胞汇合;VSMC平滑肌标志物calponin和a-SMA均呈阳性表达(P0.05);VSMC可扩增代数有限,扩增7代后即表现出细胞形态改变,增殖缓慢,胞核内出现空泡,死亡细胞增加等表现。结论预消化组织块法可较快速多量获得绵羊VSMC。     

Growth characteristics of canine pathogenic viruses in MDCK cells cultured in RPMI 1640 medium without animal protein

Madin Darby canine kidney (MDCK) cells were adapted to serum-free RPMI 1640 medium and used for cultivation of canine viruses. RPMI 1640 medium was supplemented with a soybean peptone, L-glutamine and antibiotics, so that the protein concentration was less than 5 microg/ml (RPMI/SP medium). The resulting adapted MDCK-SP cells showed steady growth after the twenty-eighth passage in RPMI/SP medium (MDCK-SP cell culture). Canine distemper virus, canine parvovirus, canine adenoviruses and canine parainfluenza virus, which are the principal components of canine combined virus vaccines, grew in the MDCK-SP cell culture as efficiently as the parental MDCK cells cultured in the conventional Eagle's MEM containing fetal bovine serum. Consequently, the use of MDCK-SP cell culture can make current canine vaccine products much safer, of higher quality and at lower cost.     

Maintenance of infectivity of Trypanosoma congolense in vitro with explants of infected skin at 37 degrees C

Glossina morsitans infected with two stocks of Trypanosoma congolense were fed on rabbits and calves to produce local skin reactions containing trypanosomes. Areas of infected skin were removed from the animals and used to prepare dermal explant cultures in Eagle's MEM and RPMI 1640 culture medium, supplemented with foetal bovine serum and containing penicillin and streptomycin. Cultures were incubated at 37 °C and media were changed at 24 to 48 hour intervals to maintain pH 7·0 to 7·2. There was evidence of trypanosome multiplication in explant cultures set up in both media; one trypanosome stock was maintained equally well in both Eagle's MEM and RPMI 1640, but the other stock survived better in Eagle's MEM. Explant cultures prepared from calf tissues generally yielded more trypanosomes at 24 hours than those prepared from rabbit tissues. The numbers of parasites present near the explants at 24 hours were maintained for up to 14 to 15 days before a decline in parasite concentration occurred. The organisms retained typical blood stream trypomastigote morphology and were infective for mice for periods up to 21 days. The trypanosomes growing in primary explant cultures could not be subpassaged in culture media alone or on to monolayers of fibroblast-like cells of bovine, murine or buffalo origin. Attempts to establish primary cultures by placing infected skin explants directly on to similar monolayers were also unsuccessful.     

早孕绒毛外细胞滋养细胞的体外培养方法

目的:探讨改良人早孕绒毛外细胞滋养细胞原代分离培养方法。方法:建立改良的复合酶消化梯度离心法,分离培养绒毛外细胞滋养细胞,通过免疫细胞化学、Giemsa染色和细胞侵袭实验检测细胞纯度、数量及生物学活性(侵袭性),并与低浓度胰蛋白酶消化方法进行比较。结果:低浓度胰蛋白酶消化方法和改良的复合酶消化梯度离心法所得贴壁细胞量分别为(11.69±1.26)×104和(17.21±0.51)×104,两者比较有统计学差异(P<0.05);绒毛外细胞滋养细胞纯度分别为(63.20±7.12)%和(92.10±5.11)%,两者比较有统计学差异(P<0.05),穿膜细胞数分别为(36.10±3.28)和(66.20±5.17),两者比较有统计学差异(P<0.05),提示该实验室改良的复合酶消化梯度离心法所得细胞总量、绒毛外细胞滋养细胞纯度及数量均显著高于低浓度胰蛋白酶消化方法。结论:该实验室改良的复合酶消化梯度离心法获得绒毛外细胞滋养细胞纯度和数量显著提高。     

微载体技术高密度培养MRC-5细胞初探

目的 通过探索MRC-5细胞的微载体培养条件,以期建立MRC-5细胞的高密度培养方法.方法 用3种不同培养基（MEM、M199、DMEM）分别培养3.0×104/mL的MRC-5细胞,观察它们的细胞增殖及传代能力;在Spinner培养系统中,用1.0 g/L Cytopore 2和3.0 g/L Cytodex 3分别培养密度为3.70× 105/mL和2.98×105/mL的MRC-5细胞,观察两种载体对细胞生长代谢和细胞密度的影响,筛选出最适宜的培养基及微载体.使用5 L CelliGen310生物反应器对筛选获得的培养基及微载体进行MRC-5细胞的灌流培养初步摸索.结果 MRC-5细胞在MEM、M199、DMEM培养基中培养96 h细胞分别增殖了7.0、4.4和3.0倍;在MEM培养基中连续传代至5次以上,细胞增殖稳定,1：3传代72 h形成致密单层,而在M199和DMEM培养基中连续传代均未能获得理想的细胞形态.经96～120 h的培养后,使用1.0 g/L Cytopore 2培养的MRC-5细胞密度达到2.20× 106/mL高于使用3.0 g/L Cytodex 3的1.12×106/mL,且前者葡萄糖和乳酸代谢较后者更为活跃.首选MEM和Cytopore 2作为MRC-5细胞的灌流培养体系.在5L生物反应器中以1.0 g/L Cytopore 2和2.5LMEM培养基培养MRC-5细胞至144h,细胞密度达到1.54×106/mL.结论 初步建立的MEM Cytopore 2微载体细胞培养方法能够获得高密度、活性良好的MRC-5传代细胞.     

纳米SiO2对大鼠肺泡Ⅰ型上皮细胞毒性的影响

目的 探讨纳米SiO2的肺细胞毒性.方法 用透射电镜观察2种不同粒径纳米SiO2粒子粒径、分散性、形状;用Zeta电位粒度分析仪检测纳米粒子在纯水和含体积分数为1%胎牛血清的RPMI 1640细胞培养液中的粒径.以大鼠肺泡Ⅰ型上皮细胞R3/1为靶细胞,利用分光光度法检测浓度均为2.5、5.0、10.0、20.0 μg/ml的2种不同粒径纳米SiO2粒子在6、24和48 h引起细胞乳酸脱氢酶(LDH)活力的变化;用酶联免疫法检测24 h时2种纳米SiO2粒子对细胞蛋白质羰基化水平及巨噬细胞炎性蛋白2(MIP-2)释放的影响.结果 2种粒径纳米SiO2粒子均呈球形,大小一致,分布均匀,分散性好,平均粒子粒径分别为(43±4.2)和(68±5.7)nm;2种粒子分别在纯水和含质量分数为1%胎牛血清的RPMI 1640细胞培养液中有相似的粒径.6、24、48 h,2.5、5.0、10.0和20.0μg/ml的43和68 nm粒子引起细胞LDH活力轻微增加,与对照组相比,差异无统计学意义(P＞0.05);作用24 h后,细胞MIP-2的释放及细胞羰基化水平轻度增加,与对照组相比,差异均无统计学意义(P＞0.05).结论 在该实验条件下,2.5、5.0、10.0、20.0 μg/ml的2种纳米粒子对R3/1细胞无明显肺毒性.     

胎盘Hofbauer细胞的体外培养和鉴定

目的 通过对早孕胎盘绒毛膜Hofbauer细胞的分离,纯化和培养,寻找一种稳定、简便的,可获得较高纯度Hofbauer细胞的培养方法,以满足后续实验要求.方法 通过胰酶/胶原酶联合消化法对妊娠6～10周绒毛组织进行消化,获得单细胞悬液,根据不同细胞贴壁时间的差异,对Hofbauer细胞进行纯化,并通过免疫组织化学法对Hofbauer细胞进行鉴定.结果 获得了较高纯度的胎盘Hofbauer细胞,免疫组化法鉴定CD68阳性,细胞生长状态良好.结论 利用胰酶/胶原酶联合消化法及差异贴壁法可以获得满意的人胎盘Hofbauer细胞.     

Hepatocytes as feeder-layers for in vitro cultivation of Plasmodium falciparum blood-stages

To improve the in vitro growth of Plasmodium falciparum we attempted to cultivate its erythrocytic stages on monolayers of functionally active hepatocytes. Hepatocytes from Swiss Albino mice were isolated by perfusing the liver with a collagenase solution and were co-cultured with a liver epithelial cell type in RPMI 1640 medium supplemented with 10% human umbilical cord serum. The results show that the presence of hepatocytes improves both the multiplication rates of three strains of P. falciparum already in cultivation and the proliferation of freshly isolated strains. Of nine primary isolates tested, only three could be adapted in the standard conditions, whereas all grew readily in the presence of hepatocytes. After two to three weeks of culture with feeder cells, all the strains could be maintained continuously in standard conditions. Similar results were obtained using hepatocytes from another rodent species. Growth was also improved using the supernatant from hepatocyte cultures. No improvement resulted from the use of two human hepatoma cell lines, one rat hepatoma, human embryonic lung fibroblasts, human liver fibroblasts and rat liver epithelial cells as feeder layers. From these results it appears that better culture media can be designed and that the effect of hepatocytes is probably related to the specific functions exhibited by these cells. Hepatocytes may act either by removing toxic substances, particularly lactic acid in the Krebs and Cori cycles, and/or supplying nutrients essential to the parasite.