角膜基质载体培养的角膜缘干细胞的增龄性变化及相关性研究
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摘要
目的：通过将不同年龄兔角膜缘干细胞种植于不同年龄兔角膜基质载体上,观察同种异体角膜基质载体培养的兔角膜缘干细胞生物学特性随增龄的变化,探讨了增龄对在角膜基质载体上培养的LSCs增殖能力的影响,同时检测了角膜基质载体培养的角膜缘干细胞的相关性标记物ΔNp63、ABCG2的表达量随增龄的变化,探讨其与载体培养的干细胞老化有无关系,为组织工程化角膜种子细胞选择的成因开辟了新途径。
方法：对照实验研究。用酶消化法将不同年龄新西兰兔角膜缘组织制成细胞悬液培养,以相同的细胞密度种植于不同年龄的新西兰兔的角膜基质上,观察角膜基质载体培养的角膜缘干细胞的生长情况,原位杂交检测ABCG2、ΔNp63的表达,鉴定LSCs, IPP5.1软件分析图像,流式细胞术测细胞周期,逆转录聚合酶链反应(RT-PCR)技术检测角膜基质载体培养的细胞中ΔNp63、ABCG2蛋白的表达量,运用SPSS13.0软件对析因设计资料进行方差分析。
结果：种植于不同年龄角膜基质载体上的青年角膜缘干细胞较其他组增殖快,潜伏期短,形态较小,以圆形、椭圆形和多边形为主,形成的集落较大。传代培养于角膜基质载体上的LSCs经ABCG2、p63原位杂交检测,DAB染色大部分细胞呈阳性反应,细胞胞浆着色呈棕黄色,胞核为蓝色。角膜缘干细胞供体年龄各水平差异有统计学意义,相同时间内角膜基质载体培养的青年兔角膜缘干细胞增殖期细胞所占比例大,ΔNp63、ABCG2表达量高；而角膜基质供体年龄各水平差异与角膜缘干细胞和角膜基质的交互作用均无统计学意义。
结论：角膜基质载体培养的角膜缘干细胞的增殖能力随着角膜缘干细胞供体年龄的增加而降低。ΔNp63、ABCG2蛋白的表达具有明显的年龄相关性,随着增龄ΔNp63、ABCG2蛋白表达量下调可能是载体培养的角膜缘干细胞老化的分子机制之一
Objectives By culturaling different ages rabbits LSCs in different age rabbits corneal stroma carrier to observe the change of aging on the biological characteristics of LSCs seeded in allogeneic corneal stroma, to investigate the effects of aging on proliferation capacity of limbal stem cells seeded in corneal stroma carrier; by detecting the effects of aging of expression ofΔNp63 and ABCG2 protein of LSCs seeded in corneal stroma to investigate the relevancy with aging of stem cell cultured in carrier, and to develop new way for corneal seeds cell selection of tissue engineering.
Methods Experimental study on the control. Limbal epithelium harvested from New Zealand rabbits of different ages were enzymatically cleft into single cell suspension and cultured, and limbal stem cells were seeded at a same cell density in the corneal stroma of different age of New Zealand white rabbits. The growth status of LSCs seeded in the corneal stroma was observed while identification was performed using in situ hybridization, image was analyzed in IPP5.1 software, cell cycle was examined by flow cytometry, the expression ofΔNp63 and ABCG2 was assessed by RT-PCR technique and the experimental data was analysed by ANOVA of factorial design.
Results The young LSCs seeded in different age corneal stroma proliferates faster than other group, the delitescence is short, smaller shape, round, ellipse, polygon, and form a larger colony. The serial subcultivation LSCs in corneal stroma carrier is identificated by ABCG2 and P63 in situ hybridization, most cells present positive reaction, the endochylema presents buffy and the nucelus presents blue. The difference in the level of LSCs donor age has statistical significance, as the ratio of proliferating cells and and the expression ofΔNp63 and ABCG2 protein was the highest in the young rabbits compared with the middle-aged and elder rabbits; the difference in the level of corneal stroma donor age and the interation of LSCs and corneal stroma has no statistical significance.
Conclusions The proliferation capacity of limbal stem cells seeded in allogeneic corneal stroma was shown to decrease with aging of the donor of limbal stem cells. The expression ofΔNp63 and ABCG2 protein decreases with aging of the donor of limbal stem cells, which suggests thatΔNp63 and ABCG2 protein may play an important role in the aging process of limbal stem cell cultured by cornea stroma.
方法：对照实验研究。用酶消化法将不同年龄新西兰兔角膜缘组织制成细胞悬液培养,以相同的细胞密度种植于不同年龄的新西兰兔的角膜基质上,观察角膜基质载体培养的角膜缘干细胞的生长情况,原位杂交检测ABCG2、ΔNp63的表达,鉴定LSCs, IPP5.1软件分析图像,流式细胞术测细胞周期,逆转录聚合酶链反应(RT-PCR)技术检测角膜基质载体培养的细胞中ΔNp63、ABCG2蛋白的表达量,运用SPSS13.0软件对析因设计资料进行方差分析。
结果：种植于不同年龄角膜基质载体上的青年角膜缘干细胞较其他组增殖快,潜伏期短,形态较小,以圆形、椭圆形和多边形为主,形成的集落较大。传代培养于角膜基质载体上的LSCs经ABCG2、p63原位杂交检测,DAB染色大部分细胞呈阳性反应,细胞胞浆着色呈棕黄色,胞核为蓝色。角膜缘干细胞供体年龄各水平差异有统计学意义,相同时间内角膜基质载体培养的青年兔角膜缘干细胞增殖期细胞所占比例大,ΔNp63、ABCG2表达量高；而角膜基质供体年龄各水平差异与角膜缘干细胞和角膜基质的交互作用均无统计学意义。
结论：角膜基质载体培养的角膜缘干细胞的增殖能力随着角膜缘干细胞供体年龄的增加而降低。ΔNp63、ABCG2蛋白的表达具有明显的年龄相关性,随着增龄ΔNp63、ABCG2蛋白表达量下调可能是载体培养的角膜缘干细胞老化的分子机制之一
Objectives By culturaling different ages rabbits LSCs in different age rabbits corneal stroma carrier to observe the change of aging on the biological characteristics of LSCs seeded in allogeneic corneal stroma, to investigate the effects of aging on proliferation capacity of limbal stem cells seeded in corneal stroma carrier; by detecting the effects of aging of expression ofΔNp63 and ABCG2 protein of LSCs seeded in corneal stroma to investigate the relevancy with aging of stem cell cultured in carrier, and to develop new way for corneal seeds cell selection of tissue engineering.
Methods Experimental study on the control. Limbal epithelium harvested from New Zealand rabbits of different ages were enzymatically cleft into single cell suspension and cultured, and limbal stem cells were seeded at a same cell density in the corneal stroma of different age of New Zealand white rabbits. The growth status of LSCs seeded in the corneal stroma was observed while identification was performed using in situ hybridization, image was analyzed in IPP5.1 software, cell cycle was examined by flow cytometry, the expression ofΔNp63 and ABCG2 was assessed by RT-PCR technique and the experimental data was analysed by ANOVA of factorial design.
Results The young LSCs seeded in different age corneal stroma proliferates faster than other group, the delitescence is short, smaller shape, round, ellipse, polygon, and form a larger colony. The serial subcultivation LSCs in corneal stroma carrier is identificated by ABCG2 and P63 in situ hybridization, most cells present positive reaction, the endochylema presents buffy and the nucelus presents blue. The difference in the level of LSCs donor age has statistical significance, as the ratio of proliferating cells and and the expression ofΔNp63 and ABCG2 protein was the highest in the young rabbits compared with the middle-aged and elder rabbits; the difference in the level of corneal stroma donor age and the interation of LSCs and corneal stroma has no statistical significance.
Conclusions The proliferation capacity of limbal stem cells seeded in allogeneic corneal stroma was shown to decrease with aging of the donor of limbal stem cells. The expression ofΔNp63 and ABCG2 protein decreases with aging of the donor of limbal stem cells, which suggests thatΔNp63 and ABCG2 protein may play an important role in the aging process of limbal stem cell cultured by cornea stroma.
引文
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