体外培养人胎儿软骨细胞的生物学特性

目的 建立人胎儿软骨细胞分离培养、扩增技术体系,研究其体外单层培养条件下的生长特性,探讨胎儿软骨细胞作为软骨组织工程种子细胞的可行性。方法酶消化法获取胎儿软骨细胞,体外培养扩增,经测定细胞生长曲线、群体倍增时间及累计倍增数目,观察细胞生长动力学并确定体外生长规律。通过免疫组织化学检测Ⅱ型胶原分泌,阿利辛蓝比色法测定基质糖胺多糖(GAG)含量。P1细胞接种于聚羟乙酸(PGA)纤维支架,观察两者生物相容性及细胞体外成软骨能力。结果 关节软骨细胞形态呈典型的多角形,随着传代的次数增加,逐渐变成“成纤维细胞样”的条梭形。平均倍增时间为74 h。细胞连续培养5代,累计倍增数目为7.83±0.42。P1-P5间接免疫组化Ⅱ型胶原表达阳性,P6不表达。GAG含量P1细胞低于P0细胞,随体外培养时间延长逐渐增加。P1细胞接种于PGA纤维支架,体外培养14 d,组织学观察显示有软骨组织形成。结论 胎儿软骨细胞经常规体外培养,至第6代失去分泌软骨特异性基质的功能。P1细胞接种PGA纤维展现良好的生物相容性,初步证实胎儿软骨细胞作为软骨组织工程种子细胞的可行性。     

人胎关节软骨细胞体外培养的生物学特性

目的：研究软骨组织工程中传软软骨细胞与原代的差异，方法：用5个月人胎关节软骨分离培养细胞，观察细胞存活率，贴壁率，生长曲线和组织形态学的改变。结果：（1）软骨块在4℃下，3天内细胞存活率可达93．4％－97．6％。（2）原代细胞为圆形或三角形；第4代有一半转变成梭形，到第6代全部变为长梭形。（3）传代细胞贴壁时间（2－3h)短于原代（4－7h)。玻璃瓶内贴壁率传代细胞为78．7％－85．5％，原代8．8％。（4）生长曲线表明，从原代到第4代都有高增殖力；到第8代时增殖降低；到第12代几乎丧失细胞增殖。结论：软骨块4度冷藏，3天内对细胞存活率无明显影响。第4代细胞壁壁快，增殖 ，适于作为组织工程用细胞，第8代以后不适合。     

体外培养大鼠耻骨联合软骨细胞的生物学特性

[目的]通过对大鼠耻骨联合软骨细胞的分离及体外培养,观察其生长、表型等一般生物学特点.[方法]采用胰蛋白酶、Ⅱ型胶原酶序贯消化法,从SD大鼠耻骨联合软骨组织中分离出软骨细胞,用含150mL/L胎牛血清的DMEM/F-12培养液原代和传代培养.分别以2g/L胶原酶消化2、6、12 h,分析不同消化时间获取的细胞数量及生长情况.对6 h消化组细胞进行Ⅱ型胶原免疫组化染色,并观察第1、4、7代的生长曲线.[结果]经胶原酶消化2、6、12 h后,平均每100mg耻骨联合软骨获取的原代细胞数之间有统计学显著差异（P＜0.01）.消化6 h组可以获取数量丰富的原代细胞,生长状态好.大鼠耻骨联合细胞原代呈短梭形、三角形或多角形,有短细胞突起.Ⅱ型胶原免疫组化染色在第1、4代培养细胞均呈阳性,第7代培养细胞呈阴性.第7代细胞生长增殖比第1、4代慢.[结论]耻骨联合软骨细胞体外培养时属于有限细胞系.培养初期（第4代之前,含第4代）的细胞较好地保持了软骨细胞的表型,且增殖能力强,有可能作为软骨组织工程的种子细胞.     

体外培养大鼠耻骨联合软骨细胞的生物学特性

 [目的]通过对大鼠耻骨联合软骨细胞的分离及体外培养,观察其生长、表型等一般生物学特点.[方法]采用胰蛋白酶、Ⅱ型胶原酶序贯消化法,从SD大鼠耻骨联合软骨组织中分离出软骨细胞,用含150mL/L胎牛血清的DMEM/F-12培养液原代和传代培养.分别以2g/L胶原酶消化2、6、12 h,分析不同消化时间获取的细胞数量及生长情况.对6 h消化组细胞进行Ⅱ型胶原免疫组化染色,并观察第1、4、7代的生长曲线.[结果]经胶原酶消化2、6、12 h后,平均每100mg耻骨联合软骨获取的原代细胞数之间有统计学显著差异（P＜0.01）.消化6 h组可以获取数量丰富的原代细胞,生长状态好.大鼠耻骨联合细胞原代呈短梭形、三角形或多角形,有短细胞突起.Ⅱ型胶原免疫组化染色在第1、4代培养细胞均呈阳性,第7代培养细胞呈阴性.第7代细胞生长增殖比第1、4代慢.[结论]耻骨联合软骨细胞体外培养时属于有限细胞系.培养初期（第4代之前,含第4代）的细胞较好地保持了软骨细胞的表型,且增殖能力强,有可能作为软骨组织工程的种子细胞.     

不同培养时间软骨细胞的生物学特性

目的 探讨软骨细胞在体外不同培养时间的生物学特性。方法 把罗骨细胞置盖玻片上２,观察不同培养时间细胞的形态学变化和增殖能力,应用阿新蓝和三色染色,观察细胞在培养过程中酸性粘多糖（ＧＡＧ）、和胶原分泌情况。结果 软骨细胞经体外单层培养,传代４～５代后,细胞形态逐渐由多角形变为俊形,基质分泌减少,ＧＡＧ和胶原染色变浅。结论 软骨细胞在体外培养３周左右是作为有组织工程的最佳种子细胞。     

冻存复苏培养的关节软骨细胞生物学特性

关节软骨（细胞）的同种异体移植是解决关节软骨损伤后修复问题的理想方法．其成功的先决条件要求细胞能够耐受低温冷冻保存并保持关节软骨细胞的生物学特性。为此，该实验在成功地建立了关节软骨细胞库的基础上，进行冻存后的关节软骨细胞培养，同时利用显微缩时录像技术，借助组织学、组织化学和分子生物学等方法．观察细胞的增殖、糖胶聚糖的合成以及Ⅱ型胶原的基因表达情况。结果表明，分离的关节软骨细胞经深低温冷冻保存后复苏．细胞在14d的培养中仍可保持关节软骨细胞所特有的形态以及对糖胺聚糖和Ⅱ型胶原的合成能力。     

人骨髓基质干细胞体外培养的生物学特性

目的　探讨在骨组织工程研究中作为种子细胞来源之一的骨髓基质干细胞体外培养后的生物学特性 .方法　获取人胎儿骨髓于含 10 0 m L· L- 1 胎牛血清的 DMEM培养液中培养 ;将骨髓基质干细胞向成骨细胞方向进行诱导 ,2～ 3wk后行钙化结节 Vonkossa和碱性磷酸酶 (AL P)钙钴染色 ;同时绘制诱导前后细胞的生长曲线、测定细胞内 AL P含量变化 ,并做统计学分析 .结果　骨髓基质干细胞经诱导后增殖速度变慢 ,但合成 AL P的能力明显增强 (P<0 .0 5 ) ;Vonkos-sa和 AL P染色呈阳性 .结论　骨髓基质干细胞取材方便 ,易于诱导为成骨细胞 ,作为骨组织工程的种子细胞应用前景良好 .     

SV40LTAg基因永生化软骨细胞体外培养的生物学特性

目的 探讨永生化关节软骨细胞体外培养的生物学特性。方法 用SV40LTAg(类人猿40病毒大T抗原)基因转染原代培养的关节软骨细胞。构建永生化关节软骨细胞系(immortalized cartilage chondrocytes)并体外培养，以正常关节软骨细胞(normal cartilage chondrocytes)作对照。倒置显微镜下观察永生化软骨细胞的形态学变化和增殖情况；应用甲苯胺蓝、番红O及Ⅱ型胶原免疫组织化学染色，观察永生化软骨细胞在培养过程中酸性粘多糖(GAG)和胶原分泌情况。结果 永生化关节软骨细胞体外培养呈贴壁单层生长，长期传代(50代)仍有很强的增殖能力，形态呈现多角形和三角形，GAG和胶原染色均呈阳性。结论 构建的永生化关节软骨细胞在体外培养能长期维持软骨细胞的特征性表型。     

软骨细胞培养及其冻存复苏后的生物学特性比较

目的 了解冻存复苏过程对保持软骨细胞生物学特性的影响。方法 对冻存1周组、1个月组、2个月组和新鲜制备组的软骨细胞进行倒置相差显微镜动态观察、特殊染色以及透射电镜等观察，在其形态和功能方面进行比较。结果 各组间软骨细胞的生长过程、特殊染色及透射电镜观察的结果差异均无显著性，但随冻存时间的延长，其存活率有所下降。结论 冻存软骨细胞与新鲜制备的软骨细胞相比保持了一些相似的生物行为，但冻存时间长短对冻存的软骨细胞存活率有影响。     

猪耳廓软骨细胞体外培养

目的 为组织工程化软骨修复机体软骨缺损提供实验基础。方法 用消化组织块培养法体外培养猪耳廓软骨细胞。结果 软骨细胞在ＰＨ值为７．２０,含１０％胎牛血清的Ｆ１２培养基中生长良好,可传代４～５代,细胞数目扩增为原来的１０～２０倍。结论 消化组织块方法培养软骨细胞是一种可以扩增软骨细胞数目的确实可行的方法。     

正常人黑素细胞体外培养及生物学鉴定

目的 体外分离、培养正常人表皮中的黑素细胞并对其进行生物学鉴定.方法 在体外黑素细胞常规培养基中以TPA/bFGF/IBMX为基本添加剂,用胰蛋白酶消化法和G418从正常人表皮中纯化黑素细胞,用多巴(Dopa)染色、黑素含量测定、酪氨酸酶活性测定、免疫组化染色等方法对体外培养的黑素细胞的形态、结构和功能进行观察.结果 多巴染色、黑素含量测定、酪氨酸酶活性测定及免疫组化染色结果均表明培养黑素细胞的生物学功能正常,保持正常的生物学特性.结论 用TPA/bFGF/IBMX作为添加剂可在体外进行正常黑素细胞的纯培养,体外培养的黑素细胞的结构和功能保持正常的生物学特性.     

HBV体外感染人胎肝细胞的鉴定方法研究

目的通过HBV体外感染22周龄原代培养人胎肝细胞,比较常用检测指标的敏感性和特异性.方法利用HBV阳性血清感染体外培养的原代人胎肝细胞.应用ELISA法检测培养上清中的HBsAg和HBeAg,免疫组化法检测细胞核中的HBcAg,荧光定量PCR法检测上清和细胞中HBV DNA,巢式PCR法检测细胞中cccDNA.结果胎肝细胞核中的HBcAg于感染后第2天出现阳性,培养上清和细胞中HBVDNA定量自第4天起检出,上清中HBsAg和HBeAg于第5～6天出现阳性;细胞中HBV cccDNA自第8～9天出现阳性.结论HBV在原代培养人22周龄的胎肝细胞中能够稳定复制和表达,各种检测指标的灵敏性和特异性不一,免疫组化法检测细胞核中的HBcAg敏感性好,特异性可.     

Parthenolide inhibits tumor necrosis factor-alpha induced catabolism of aggrecan in chondrocytes in osteoarthritis: in vitro experiment with cultured human chondrocytes

OBJECTIVE: To investigate the effect of parthenolide (PAR) on the tumor necrosis factor (TNF)-alpha induced aggrecan catabolism of chondrocytes in ostroarthritis (OA). METHODS: Human chondrocytes were obtained from the condyles of femur of OA patients undergoing knee joint replacement during operation, cultured, and randomly divided into 4 groups: control group, TNF group (cultured in medium containing 10 ng/ml TNF-alpha) , PAR group (cultured in medium containing 10 micromol/L PAR), and PAR + TNF group (cultured in medium containing 10 micromol/L PAR and 10 ng/ml TNF-alpha). Eight days later 1, 9-dimethylmethylene blue spectrophotometric assay was used to measure the content of glycosaminoglycan (GAG) , marker of aggrecan catabolism, in the culture fluid and the cells. Using anti-aggrecan monoclonal antibodies Mab 5D4 and 3B3, ELISA was employed to detect the contents of 5D4 and 3B3, aggrecan catabolic fragments. RT-PCR was used to detect the mRNA expression of the aggrecan, ADAMTS-4 and ADAMTS-5, both aggrecanases, tissue inhibitor of metalloprotease (TIMP)-1, mitogen-activated protein kinase (MAPK)-1 and 18S, a marker. RESULTS: The GAG percentage in the culture fluid of the TNF-alpha was 57.1% +/- 2.0%, significantly higher than those of the control and TNF-alpha + PAR groups (P = 0.001 and 0.02). The 5D4 fragment level of the TNF-alpha group was 509 ng/ml +/- 32 ng/ml, significantly higher than that of the control group (166 ng/ml +/- 15 ng/ml, t = 11.60, P =0.007), and the level of 5D4 fragment of the PAR + TNF-alpha group was 333 ng/ml +/- 15 ng/ml, significantly lower than that of the TNF-alpha group (t = 7.93, P = 0.016). There was not significant difference in the 3B3 fragment level among the 4 groups (F = 1.316, P = 0.335). The aggrecan mRNA expression level of the TNF-alpha group was significantly lower than those of the other 3 groups (F = 133.7, P = 0.000), the mRNA expression levels of ADAMTS-5 and MAPK-1 of the TNF-alpha group were significantly higher than those of the other 3 groups (F = 209. 7, 117.1; P =0. 000), the ADAMTS-5 mRNA expression level of the PAR group was significantly lower than that of the control group (t = 11.1, P= 0.008) , and there was not significant differences in the mRNA expression levels of ADAMTS-4 and TIMP-1 among the 4 groups (F = 1.87, 0.73; P > 0.05) . CONCLUSION: PAR inhibits TNF-alpha induced catabolism of aggrecan in the chondrocytes of OA and reduces the mRNA expression of ADAMTS-5 and MAPK-1. PAR may be useful in the treatment of OA and other inflammatory joint diseases.     

肿瘤坏死因子-α对软骨细胞蛋白聚糖代谢的影响及小白菊内酯的保护作用

目的 探讨小白菊内酯(PAR)对关节软骨细胞蛋白聚糖合成的调节作用.方法 软骨细胞取自骨关节炎(OA)患者进行膝关节置换术的股骨髁,随机分为4组:对照组、肿瘤坏死因子-α(TNF-α)组、PAR组和TNF-α+PAR组.用3种方法测定蛋白聚糖的代谢:(1)测定蛋白聚糖的代谢产物糖胺多糖(GAG)含量.(2)用针对蛋白聚糖单克隆抗体(Mab-383和5D4),采用ELISA法检测培养液中蛋白聚糖的代谢片段含量.(3)半定量RT-PCR检测软骨细胞蛋白聚糖、ADAMTS-4、ADAMTS-5、基质金属蛋白酶组织抑制因子-1(TIMP-1)和丝裂原活化蛋白激酶.1(MAPK-1)的mRNA 表达.结果 (1)TNF-α+PAR组培养液中GAG的百分含量明显低于TNF-α组(t=5.92,P=0.02),其降低程度与PAR呈剂量依赖性.(2)TNF-α组培养液中5D4片段的含量明显高于TNF-α+PAR组(t=7.93,P=0.016),而各组间培养液中383片段的含量无差别.(3)TNF-α组的蛋白聚糖mRNA表达明显低于其他3组(F=133.7,P=0.000);而其ADAMTS-5及MAPK-1的mRNA表达明显高于其他3组(F=209.7,117.1;P=0.000).结论 (1)TNF-α可促进人OA关节软骨细胞蛋白聚糖的降解而PAR可抑制其降解作用.(2)PAR可下调TNF-α导致的ADAMTS-5及MAPK-1的mRNA表达.     

骺板软骨细胞复合生物凝胶体外培养生成软骨组织的实验研究

目的 观察将骺板软骨细胞接种于自制的生物凝胶经体外培养生成软骨组织的生物学特点，初步评价这一凝胶基质作为软骨组织构建材料的生物学性能。方法 将第一代骺板软骨细胞接种于生物凝胶进行体外培养，行大体，倒置显微镜以及组织学，Ⅰ型和Ⅱ型胶原免疫组化光镜观察。结果 骺板软骨细胞－生物凝胶复合的，在体外培养过程中不能春初始外形，培养1周直径可收缩为初始的45％，但能保持种子细胞的稳定均发布。经体外培养2周后由外周向中心逐渐形成软骨组织，表面为由1－3层梭形细胞组成的膜样结构，内部细胞主要为圆形细胞，有细胞外基质相隔，形成软骨细胞陷窝。基质中富含软骨特异性基质成分Ⅱ型胶原和蛋白聚糖，而Ⅰ型胶原免疫组化逐渐转为弱阳性，位于表面膜结构。结论 这一生物凝胶具有良好的细胞相容性，适合软骨细胞在其中生成成熟的工程化软骨，但难以维持其初始外形。     

骨髓间充质干细胞在软骨细胞培养条件下生物学特点的观察

目的 观察骨髓间充质干细胞（Mesenchymal stem cells,MSCs)在软骨细胞培养条件下传代培养的主要生物学特性。方法 梯度离心分离幼兔骨髓有核细胞，培养分离MSCs并进行传代培养，观测在体外软骨细胞培养条件下MSCs形态，生长特点及Ⅰ型和Ⅱ型胶原，蛋白多糖聚集体合成分泌以及碱性磷酸酶活性等方面的变化。结果 （1）MSCs原代细胞呈可见的均匀分布的簇状生长，呈长梭形，在传代培养中形态特性无明显变化，均质性明显提高。（2）各代Ⅰ型胶原免疫组化均为阳性，Ⅱ型胶原免疫组化均为阴性。（3）在各代培养中碱性磷酸酶染色均为阴性，对甲苯胺蓝存在极弱的异染性反应；（4）细胞外基质中硫酸糖胺多糖含量亦很低。结论 传代培养中骨髓MSCs保持了常规低糖培养条件下的基本生物学特点，说明常用的软骨细胞基础培养条件同样适合骨髓MSCs的培养。     

Biological characterization of cultured dermal papilla cells and hair follicle regeneration in vitro and in vivo

Background Dermal papilla cells (DPC) are a group of mesenchyme-derived cells at the base of the hair follicle, where they regulate and control hair follicle growth through the expression and secretion of cytokines. Nevertheless, the role of DPC derived chemokines and other cytokines in the hair follicle biology remain speculative. In this study, we investigated the expression of basic fibroblast growth factor (bFGF), endothelin-1 (ET-1) and stem cell factor (SCF) in different passages of cultured DPC and their effects on the biological behaviour of DPC.Methods The expression of bFGF, ET-1 and SCF in different passages of cultured DPC and their possible effects on the biological behavior of DPC are investigated using in situ hybridization and immunochemistry. In addition, we performed transplantation of hair follicle cells into nude mice. The cultured DPC, dermal sheath cells and fibroblast of human scalp, respectively, were mixed with cells of the hair follicle epithelium in different ratios, and then were cultured in hair follicle organotypic cultures or implanted into the subcutis of nude mice.Results The expression of ET-1 and SCF in early passages of cultured DPC became stronger, but turned weaker and even negative in late passages (>6 passages). Hair follicle-like structures were formed after DPC combined with the cells of hair follicle epithelium cells in hair follicle organotypic cultures. When hair follicle organotypic cultures were implanted into the subcutis of nude mice, the relative intact hair follicles were formed. After the transplantation of hair follicle cells into the nude mice, the hair follicle-like structure was formed in the group that contained DPC mixed with hair follicle epithelium cells. However, no hair follicles were formed in the other two groups. It was found that the higher the expression of ET-1 and SCF in DPC, the stronger the ability of DPC to induce hair follicle regeneration.Conclusions The cultured DPC can induce hair follicle regeneration and sustain hair growth in vivo and in vitro. Moreover, the expression of ET-1 and SCF is correlated with the ability of DPC inducing hair follicle regeneration.     

Biological characterization of cultured dermal papilla cells and hair follicle regeneration in vitro and in vivo

Background Dermal papilla cells （DPC） are a group of mesenchyme-derived cells at the base of the hair follicle, where they regulate and control hair follicle growth through the expression and secretion of cytokines. Nevertheless, the role of DPC derived chemokines and other cytokines in the hair follicle biology remain speculative. In this study, we investigated the expression of basic fibroblast growth factor （bFGF）, endothelin-1 （ET-1） and stem cell factor （SCF） in different passages of cultured DPC and their effects on the biological behaviour of DPC. Methods The expression of bFGF, ET-1 and SCF in different passages of cultured DPC and their possible effects on the biological behavior of DPC are investigated using in sire hybridization and immunochemistry. In addition, we performed transplantation of hair follicle cells into nude mice. The cultured DPC, dermal sheath cells and fibroblast of human scalp, respectively, were mixed with cells of the hair follicle epithelium in different ratios, and then were cultured in hair follicle organotypic cultures or implanted into the subcutis of nude mice. Results The expression of ET-1 and SCF in early passages of cultured DPC became stronger, but turned weaker and even negative in late passages （〉6 passages）. Hair follicle-like structures were formed after DPC combined with the cells of hair follicle epithelium cells in hair follicle organotypic cultures. When hair follicle organotypic cultures were implanted into the subcutis of nude mice, the relative intact hair follicles were formed. After the transplantation of hair follicle cells into the nude mice, the hair follicle-like structure was formed in the group that contained DPC mixed with hair follicle epithelium cells. However, no hair follicles were formed in the other two groups. It was found that the higher the expression of ET-1 and SCF in DPC, the stronger the ability of DPC to induce hair follicle regeneration. Conclusions The cultured DPC can induce hair follicle regeneration and sustain hair growth in vivo and in vitro. Moreover, the expression of ET-1 and SCF is correlated with the ability of DPC inducing hair follicle regeneration.