ABSTRACT
Objective:To construct a TPC-1 cell model that stably knocks out the HMGA2 by using CRISPR/Cas9 gene editing technology. Methods:Recombinant pLV[2gRNA]-EGFP:T2A:Puro- U6> {hHMGA2 [gRNA# A1]*}- U6>{hHMGA2 [gRNA#A2]*} of lentiviral plasmid vector was constructed: targeting HMGA2 Dual-gRNA sequence was designed, the synthesized Dual-gRNA fragment into pLV [2gRNA]-EGFP was cloned: T2A:Puro-U6 vector, extract a single clone for sequencing verification. the constructed recombinant plasmid vector with lentivirus was packed, and TPC-1 cells were infected, puromycin was used to obtain HMGA2 knock-out single clone, PCR and sequencing verification were performed, and real-time fluorescent quantitative qPCR was used to detect HMGA2 mRNA in cells Knockout efficiency. Results:After sequencing verification, pLV [2gRNA]-EGFP targeting HMGA2: T2A: Puro-U6>{hHMGA2 [gRNA#A1]*}-U6>{hHMGA2 [gRNA #A2]*} plasmid was successfully constructed; A single clone was picked for PCR identification and gene sequencing, TPC-1 cells were successfully obtained with HMGA2 gene completely knocked out; TPC-1 cells with HMGA2 knocked out were detected by real-time fluorescent quantitative qPCR, and they did not express HMGA2 mRNA.Conclusion:CRISPR/Cas9 gene editing technology enables us to construct a human papillary thyroid cancer cell line TPC-1 cell model with stable knockout of HMGA2.
ABSTRACT
Objective To investigate the relationship between apoptotic protein Bcl-2 and Bax in rat hippocampal neurons and cognitive dysfunction in rats with abnormal thyroid function.Methods Thirty healthy Wistar rats of 8-week-old SPF grade were randomly divided into three groups:(1) Normal control group (n=10);(2)hypothyroidism group (n=10);(3) hyperthyroidism group (n=10).All rats were sacrificed at the 4th week by heart blood sampling.The serum T3 and T4,TSH were measured.Morris water maze was used to train rats in each group for 6 days.At the end of the experiment,the hippocampus was taken from the rats,and HE staining was performed.The expression of apoptotic protein bcl-2 and Bax in rat hippocampal neurons was detected by immunohistochemistry.Results ①The escape latency of hyperthyroidism and hypothyroidism group was higher than that of the normal group at different time points (P＜0.05).In the test of the target area dwell time,the difference between the hyperthyroid group,the hypothyroid group and the normal group was statistically significant (P＜0.05).In the distance test of the target quadrant,The differences between the three groups were statistically significant (P＜0.05).In the number of passes through the target quadrant,the difference between hyperthyroidism group,hypothyroidism group and normal group was statistically significant (P＜0.05),but there is no significant difference between hyperthyroidism group and hypothyroid group (P＞0.05).②Hippocampus tissue HE staining:normal control group hippocampal neurons neatly arranged,the shape of the structure was complete and uniform,the nucleus was round or oval,nucleolus obvious,chromatin uniform level and more clear,nucleus round or oval,obviously,hyperthyroidism group,hypothyroidism group of neuronal structure loose,the number decreased,arranged disorder,deep nuclear staining,shrinkage,nucleolus disappeared,cytoplasm around the empty halo,cell spacing larger.③The positive cells expressing Bcl-2 and Bax protein in the hyperthyroidism group and the hypothyroidism group were increased compared with the normal control group (P＜0.05).Compareds with hyperthyroidism group,the expression of Bcl-2 positive cells was increased in hypothyroidism (P＜0.05).Conclusions ①The spatial learning and memory abilities of the rats with hypothyroidism and the hyperthyroid are lower than those in the normal control group.②The number of apoptotic protein positive cells in Bcl-2 and Bax neurons of hippocampus in rats with hypothyroidism and hyperthyroid increased,and the proportion of Bcl-2 and Bax was impaired,which indicates that apoptosis occurred in hippocampal neurons.This process may be one of the pathogenesis of cognitive impairment.