The Preliminary Study On The Expression Of NKx6.1 Affected By Thyroid Hormone In Primary Cultured Astrocytes

Objective: Thyroid hormone(TH) plays a key role in the development of brain, which has a correlation with homeobox gene Nkxs. Our experiments have demonstrated Nkxs involved brain retardation caused by low TH, but the specific mechanism is unclear. According to the literature, TH, Nkx6.1 and Shh signaling pathway have certain connection. Therefore, this experiment selected Nkx6.1, Shh as research subjects, select the highest number of astrocytes in the central nervous system as the carrier.This paper aims to observe whether Shh is involved in the role of thyroid hormone affects the expression of Nkx6.1,lay the foundation for further study on thyroid hormone affects the expression of Nkx6.1.Methods: This experiment using the classical method to isolate the rat cerebral cortex to obtaine astrocytes, the method of differential velocity adherent and shaking table at constant temperature were purified on cell,cell morphology and number changes were observed with the inverted microscope, using cell immunofluorescence to observe astrocytes’ identification and purity, normal cell culture. Taking the second generation cells which were divided into three groups(low hormone group, normal group, high hormone group), in each group were cultured with 12 h, 24 h, 48 h,and then cells were collected, total RNA were extracted purity identification. Primer design, quantitative PCR to detecte the changes of different time points,and in the levels of different hormones of Shh, NKx6.1, m RNA, the preparation of protein sample, Western Blot comparison of Shh, NKx6.1 protein expression. using the endogenous Shh inhibitor Cyclopamine to culture cells 12 h, 24 h, 48 h, quantitative PCR, detecting the expression of NKx6.1 to use Western Blot at different time points.Results: The primary culture cells were placed under the inverted microscope, astrocytes after primary 24 h in planting, most of the cells have attached to the culture bottle bottom, part of the cells began to spread, the cells were round, high refraction, cells cultured in small volume,after 4-5d of culture, cell volume increased obviously, part of cells which were growing rapidly, the cell body stretching, along with the prolongation of the culture time, the numbe of cells increased obviously, the cell body increased, the ratio of astrocyte culture gradually increased at the bottom. Culture of 9-12 d,the cells basic covered the culture bottle and mainly were astrocytes, cell arranged densely growth, cell volume reached the maximum. The cells were passaged the second time,and growed more active, cultivation of 4-6d, the bottom of the bottle can be covered fully. Cells were purified and then cultured, astrocytes form more prominent and a higher proportion. Cells were identified by GFAP immunofluorescence method, each coverslip take ten vision for positive cell counts, labeled positive antibody of astrocytes purity are more than 95%. Real-time quantitative PCR Western Blot to detect the change of Shh, NKx6.1, the control group showed that in the measured time Nkx6.1 have no obvious changes, just a small expression peak in 48h; in the low hormone group,in 12 h, 24 h culture time, Nkx6.1 did not change significantly, and compared with the normal control group, the difference was not significant(P > 0.05), in 48 h amount of expression,there was a sudden drop;in the high hormone group, after the culture of 12 h, 24 h, Nkx6.1 levels increased significantly, the amount of expression in 48 h decreased suddenly and was significantly lower than that of 12 h, 24h(P < 0.05). On the other hand, in the control group, the level of Shh in the measured time have no obvious change; in the low hormone group, levels of Shh were significantly decreased in 12 h, 24 h, 48 h, and a maximum decline in 48h; in the high hormone group culture of 12 h, 24 h,the level of Shh increased significantly, whereas in 48 h the expression quantity reduced significantly. Study on the correlation between Shh and Nkx6.1 shows that, after the inhibition of Shh,the level of Nkx6.1 decreased with time increased, and have significant difference compared with the control group(P < 0.01).Conclusion: We have obtained successfully high purity astrocytes useing of primary culture techniques, confirmed the role of thyroid hormone in different doses at different times with different effects on Shh and Nkx6.1,Shh and Nkx6.1 has similar level changes. By further inhibit Shh signaling pathway,we found that regardless of what level of thyroid hormone, NKx6.1 expression was down-regulated. In summary,initially confirmed that Shh involves in the process of thyroid hormone on NKx6.1,and Shh is likely to be a key factor to reveal the mechanism of this process.