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使用牛垂体提取物在体外增加角膜细胞的增殖并维持其表型。

Using bovine pituitary extract to increase proliferation of keratocytes and maintain their phenotype in vitro.

作者信息

Xu Zhong-Zhong, Li Zhi-Jie, Du Lian-Xin, Li Jing, Wang Li-Ya

机构信息

Department of Ophthalmology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan Province, China ; Key Laboratory of Corneal Disease, Henan Eye Institute, Zhengzhou 450003, Henan Province, China.

Department of Pediatrics, Baylor College of Medicine, Houston 77030, Texas, USA.

出版信息

Int J Ophthalmol. 2013 Dec 18;6(6):758-65. doi: 10.3980/j.issn.2222-3959.2013.06.04. eCollection 2013.

Abstract

AIM

To investigate the effects of bovine pituitary extract on the proliferation of keratocytes and maintaining the keratocyte phenotype in vitro.

METHODS

Single keratocytes were isolated by enzyme digestion for in vitro culture. Three groups were designed according to the different culture media: a bovine pituitary extract (BPE) group, a fetal bovine serum (FBS) group and the control group. The phenotypes and proliferation of cultured cells were evaluated by morphology, immunofluorescent staining and mRNA expression of CD34, Lumican, VSX1, α-SMA and proliferating cell nuclear antigen (PCNA). In the BPE group, cells underwent serial subcultivation, and their phenotypes were identified by immunofluorescent staining. To analyze the proliferation of keratocytes in different concentrations of BPE, six different concentrations were designed to ascertain the most appropriate amount.

RESULTS

In the BPE group, the cells spread out and presented dendritic morphology, and their dendrites connected to one another to form networks. On the third passage, most cells maintained their phenotype. In the FBS group, the cells exhibited a dendritic appearance in early cultured stages, but their morphology subsequently changed into a fibroblast-like shape. The number of dendritic cells in BPE group was more than FBS and control groups. Immunofluorescent staining and real-time polymerase chain reaction (PCR) confirmed that few keratocytes underwent fibroblastic transformation in the BPE and control groups, and that proliferation was higher in the BPE group than in the control group. Although the proliferation was higher in the FBS group, many keratocytes underwent fibroblastic transformation. The analysis of cell morphology and mRNA expressions of CD34, PCNA and VSX1 in six group showed that different concentrations of BPE affected the proliferation obviously but didn't affect the keratocyte phenotype, and the concentration of 40µg/mL was the most appropriate one.

CONCLUSION

BPE can improve the proliferation of keratocytes and maintain their phenotype in vitro. Many keratocytes can be harvested rapidly and provide seeds for the construction of corneal stroma.

摘要

目的

研究牛垂体提取物对体外培养的角膜细胞增殖及维持角膜细胞表型的影响。

方法

采用酶消化法分离单个角膜细胞进行体外培养。根据不同培养基设计三组:牛垂体提取物(BPE)组、胎牛血清(FBS)组和对照组。通过形态学、免疫荧光染色以及CD34、亮氨酸聚糖、VSX1、α-平滑肌肌动蛋白(α-SMA)和增殖细胞核抗原(PCNA)的mRNA表达来评估培养细胞的表型和增殖情况。在BPE组中,细胞进行连续传代培养,并通过免疫荧光染色鉴定其表型。为分析不同浓度BPE对角膜细胞增殖的影响,设计了六种不同浓度以确定最合适的用量。

结果

在BPE组中,细胞铺展并呈现树突状形态,其树突相互连接形成网络。在第三代时,大多数细胞维持其表型。在FBS组中,细胞在培养早期呈现树突状外观,但随后形态转变为成纤维细胞样形状。BPE组中树突状细胞的数量多于FBS组和对照组。免疫荧光染色和实时聚合酶链反应(PCR)证实,BPE组和对照组中很少有角膜细胞发生成纤维细胞转化,且BPE组的增殖率高于对照组。虽然FBS组的增殖率较高,但许多角膜细胞发生了成纤维细胞转化。对六组细胞形态以及CD34、PCNA和VSX1的mRNA表达分析表明,不同浓度的BPE对增殖有明显影响,但不影响角膜细胞表型,40μg/mL的浓度最为合适。

结论

BPE可促进体外培养角膜细胞的增殖并维持其表型。可快速收获大量角膜细胞,为角膜基质构建提供种子细胞。

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