CAJ | 학술논문

背景：椎间盘为负重却缺乏血运的组织，在体外培养过程中存在表型丢失问题，因而容易发生退行性改变，但椎间盘退变的机制尚不明确。 　　目的：探讨兔髓核细胞分离、贴壁培养、扩增和鉴定的方法，并观察髓核细胞在不同代次的生长特性。 　　方法：应用Ⅱ型胶原酶消化法和组织块贴壁法相结合的方法，分离、纯化髓核细胞并进行体外扩增，用倒置显微镜观察各代细胞的形态、生长状况，计数细胞数量，并绘制细胞生长曲线。苏木精-伊红染色后光镜下观察细胞形态，免疫细胞化学方法检测细胞Ⅱ型胶原和聚集蛋白聚糖的表达情况，并进行细胞鉴定。 　　结果与结论：成功的实现了兔髓核细胞体外分离、培养及扩增。生长特性观察发现，髓核细胞第1-3代细胞增殖能力强，活力旺盛，但随着传代次数的增加，细胞增殖能力程逐渐下降的趋势。分离培养的髓核细胞阳性表达聚集蛋白聚糖和Ⅱ型胶原。体外采用Ⅱ型胶原酶消化法和组织块贴壁法相结合，可获得高纯度的髓核细胞，培养的髓核细胞呈类圆形或多角形生长，第1-3代细胞生长活性较强。
배경：추간반위부중각결핍혈운적조직，재체외배양과정중존재표형주실문제，인이용역발생퇴행성개변，단추간반퇴변적궤제상불명학。 　　목적：탐토토수핵세포분리、첩벽배양、확증화감정적방법，병관찰수핵세포재불동대차적생장특성。 　　방법：응용Ⅱ형효원매소화법화조직괴첩벽법상결합적방법，분리、순화수핵세포병진행체외확증，용도치현미경관찰각대세포적형태、생장상황，계수세포수량，병회제세포생장곡선。소목정-이홍염색후광경하관찰세포형태，면역세포화학방법검측세포Ⅱ형효원화취집단백취당적표체정황，병진행세포감정。 　　결과여결론：성공적실현료토수핵세포체외분리、배양급확증。생장특성관찰발현，수핵세포제1-3대세포증식능력강，활력왕성，단수착전대차수적증가，세포증식능력정축점하강적추세。분리배양적수핵세포양성표체취집단백취당화Ⅱ형효원。체외채용Ⅱ형효원매소화법화조직괴첩벽법상결합，가획득고순도적수핵세포，배양적수핵세포정류원형혹다각형생장，제1-3대세포생장활성교강。
BACKGROUND:Intervertebral disc can bear load but lack vessels. Nucleus pulposus cel s have the problem of phenotype loss during in vitro culture that can lead to degenerative changes. The mechanism of intervertebral disc degeneration remains unclear. OBJECTIVE:To explore the approaches of isolation, adherence culture, amplification and identification of the rabbit nucleus pulposus cel s in vitro, to observe the growth characteristics of nucleus pulposus cel s in different generations. METHODS:Type Ⅱ col agenase digestion method plus explants culture method was used to isolate and purify nucleus pulposus cel s and then amplify in vitro. The morphology and growth of primary and passaged cel s was observed under the inverted microscope, the number of cel s was counted and the growth curve was draw. The morphology of the cel s was observed under light microscope after hematoxylin-eosin staining, and the expressions of col agen type Ⅱ and aggrecan were examined by immunocytochemistry. RESULTS AND CONCLUSION:Nucleus pulposus cel s of rabbit were isolated, cultured and amplified in vitro successful y. Growth activity was observed, and found that the 1-3 generation nucleus pulposus cel s proliferated more rapidly and vigorously. The proliferation of nucleus pulposus cel s was decreased while the cel passaged more generations. These isolated and cultured nucleus pulposus cel s could positively express the col agen type Ⅱ and aggrecan. In vitro combination of type Ⅱ col agenase digestion method and explants culture method could obtain high purity nucleus pulposus cel s, and the cultured nucleus pulposus cel s were grew in round or polygonal. The 1-3 generation of cel s had strong activity.