摘要
背景:传统培养方法体外培养的软骨细胞经长时间传代培养后往往去分化为成纤维细胞,导致细胞数量及活性下降。如何避免培养过程中的去分化问题,是模拟体内软骨内成骨软骨发育过程的关键。目的:课题创新性提出以Ⅱ型胶原酶和胰蛋白酶结合消化法体外培养扩增C57BL/6小鼠关节软骨细胞并诱导其向更成熟肥大软骨细胞或终末分化软骨细胞分化。设计、时间及地点:细胞学体外观察实验,于2008-09/12在解放军第三军医大学大坪医院野战外科研究所全军战创伤中心,创伤、烧伤与复合伤国家重点实验室完成。材料:C57BL/6品系新生小鼠9只。方法:采用胰蛋白酶和Ⅱ型胶原酶结合消化法分离培养C57BL/6新生小鼠关节软骨细胞,细胞计数法绘制细胞生长曲线,RT-PCR检测Ⅱ型胶原进行鉴定。当细胞90%融合时以含5.5mg/L人转铁蛋白,3×10-8mol/L亚硒酸钠,10mg/L牛胰岛素的ITS诱导培养基进行分化诱导。主要观察指标:诱导0,7d,阿利新蓝染色检测软骨细胞分泌基质中的葡萄糖胺聚糖,vonKossa染色检测软骨细胞钙化结节形成情况,实时定量PCR检测Ⅱ型胶原、X型胶原及基质金属蛋白酶13的表达进行鉴定。结果:原代培养细胞24h贴壁,呈三角或多角形,培养4～6d进入快速增殖期,RT-PCR检测到Ⅱ型胶原的表达,证实获得大量高纯度、高活性的软骨细胞。诱导0d,细胞单层铺满培养皿底面,无聚集现象;诱导7d明显可见由软骨细胞聚集形成的软骨小结。与诱导0d时相比,诱导7d阿利新蓝染色示软骨细胞分泌基质中的葡萄糖胺聚糖染色呈阳性,可见明显的软骨小结,Ⅱ型胶原及基质金属蛋白酶13表达明显增高,且钙化结节明显增多。结论:采用胰蛋白酶和胶原酶结合消化法获得大量高纯度、高活性的软骨细胞。ITS诱导体系有效地促进了软骨细胞成熟及终末分化,较成功模拟了软骨内成骨的软骨发育过程。
BACKGROUND： Chondrocytes in vitro cultured with traditional culture methods tend to ded i fferentiate into fibroblasts through long-term serial subcultivation, which results in a terrible decrease in the number and activity of cells. Accordingly, how to avoid the dedifferentiation during culture has become the key issue of modeling the cartilage development process of in vivo entochondrostosis.
OBJECTIVE： This study proposes innovationally to in vitro culture and amplify articular chondrocytes of C57BL/6 mice through digestion by type Ⅱ collagenase combined with trypsinase, and at the same time, to induce articular chondrocytes of mouse into the more mature hypertrophic chondrocytes or terminally differentiated chondrocytes.
DESIGN, TIME AND SETTING： A cytological in vitro observational experiment was performed at the State Key Laboratory of Trauma, Burns and Combined Injury, Army Trauma Center, Institute of Field Battle Surgery Research, Daping Hospital Affiliated to the Third Military Medical University of Chinese PLA, from September to December in 2008.
MATERIALS： Nine newborn C57BL/6 mice.
METHODS： Chondrocytes were obtained from the articular cartilage of newborn C57BL/6 mice through digestion by trypsinase combined with type Ⅱ collagenases. Cell growth curve was drawn by cell counting. The chondrocytes were detected by RT-PCR to confirm the mRNA expression of type Ⅱ collagen. When reaching 90% conTluency, the chondrocytes were cultured in ITS induction medium containing 5.5 mg/L human transferring, 3×10^- 8 mol/L sodium selenite and 10 mg/L bovine insulin.
MAIN OUTCOME MEASURES： After 7 days of induction, alcian blue staining was used for determining the glycosaminoglycan in the matrix secretion of chondrocytes, von Kossa staining for measuring calcified nodule formation of chondrocytes and Real Time-PCR for detecting the expression level of type Ⅱ collagen m RNA, type X collagen and matrix metalloproteinase 13. RESULTS： The chondrocytes in primary culture were adherent after 24 hours of culture, exhibiting triangle or polygon shape. The high proliferative phase arrived at day 4 6 after culture. The detection of type Ⅱ collagen expression proved that a large quantity of chondrocytes of high purity and activity were obtained successfully. Before induction, monolayer chondrocytes scattered across the bottom of the culture disc, without any cluster. After 7 days of induction, many chondrocytes gatered into obvious cartilaginous nodes. Comparatively, chondrocytes induced for 7 days showed positive results to the alcian blue staining of glycosaminoglycan in its matrix secretion and obvious cartilaginous nodes could be seen in it. What＇s more, expression of type Ⅱ collagen and matrix metalloproteinase 13 increased obviously and so did the calcified nodules.
CONCLUSION： Large quantities of chondrocytes of high purity and activity can be harvested through digestion with type Ⅱ collagenase and trypsinase. ITS inductive system is effective in encouraging the maturity and terminal differentiation of chondrocytes. The cartilage development process in entochondrostosis is modeled successfully.
出处
《中国组织工程研究与临床康复》
CSCD
北大核心
2009年第41期8104-8108,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金重点资助项目(30530410)
国家重点基础研究发展规划项目(2005CB522604)~~
