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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.
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Objective:To investigate the risk factors and predictive effect of lateral cervical lymph node metastasis of papillary thyroid carcinoma (PTC) by applying the concept of central lymph node metastasis intensity.Methods:This study retrospectively analyzed integrated clinic data of 106 cases with PTC undergoing treatment of cervical lymph node dissection in Department of Thyroid and Breast Surgery of the Affiliated Hospital of Inner Mongolia Medical University from Dec. 2009 to Jan. 2014. Based on whether lateral cervical lymph nodes had metastasis, patents were classified into lymph node metastasis positive group ( n=75 cases) , lymph node metastasis negative group ( n=31 cases) . This study explored metastasis-associated risk factors of age, gender, triiodothyronine (T3) , thyroxine (T4) , free triiodothyronine (FT3) , free thyroxine (FT4) , thyroid stimulating hormone (TSH) , thyroglobulin antibody (TGAb) , thyroid peroxidase antibody (TPOAb) , whether combined with Hashimoto’s disease, tumor location, infringing the membrane, mulifocality, tumor glands distribution, tumor diameter, number of central lymph node metastases, central lymph node metastasis ratio, and analyzed the effects of central lymph node metastasis intensity on lateral cervical lymph node metastasis. SPSS 21.0 software was used for data analysis, the metering data of normal distribution was expressed as ± s, and t test was used for comparison between groups. Count data was expressed as a rate (composition ratio) , and comparisons between groups were performed by χ2 test or Fisher exact probability method. Results:Univariate analysis found that whether combined with Hashimoto’s disease ( P=0.087) , tumor location ( P=0.249) , tumor glands distribution ( P=0.219) and tumor diameter ( P=0.224) had no correlation with lateral cervical lymph node metastasis, which showed no statistical significant differences ( P>0.05) . Infringing the membrane ( P=0.030) , mulifocality ( P=0.031) , number of central lymph node metastases ( P=0.022) and central lymph node metastasis ratio ( P=0.001) had correlation with lateral cervical lymph node metastasis, which showed statistical significant differences ( P<0.05) . The number of central lymph node metastases and the increase of central lymph node metastasis ratio had positive correlation with the occurrence of lateral cervical lymph node metastasis; when the number of central lymph node metastases was ≥4 or (and) the central lymph node metastasis ratio was ≥20%, the incidence of lateral cervical lymph node metastases increased significantly, and the difference was statistically significant ( P<0.05) . Conclusion:Infringing the membrane and mulifocality are risk factors for lateral cervical lymph node metastasis. When central lymph node metastasis intensity: number of metastases ≥4 or (and) metastasis ratio ≥20%, lateral cervical lymph node dissection is recommended.
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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.
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Objective To investigate the effect of high serum thyroid stimulating hormone (TSH) on malignant transformation of thyroid tissue in Hashimoto's thyroiditis (HT).Methods The data of 569 patients pathologically confirmed HT admitted from Jan.2009 to Dec.2013 were retrospectively analyzed.According to with or without papillary thyroid carcinoma (PTC),patients were divided into HT group and HT complicated with PTC group.Age,gender,preoperative TSH,free thyroxine,free tri-iodothyronine,thyroidperoxidaseantibody (TPOAb),antithyroglobulinanfibody (TGAb),and tumor multifocality were investigated.Results Among the 569 patients,306 patients had HT without PTC and 263 had HT with PTC.The avaerage age of patients complicated with PTC was (45.52±10.58) years,younger than that of patients without PTC (51.63+9.50) years (P＜0.05).In HT patients with or without PTC,31％ (81/263) and 22.5％ (69/306) of patients had high TSH levels,which was significantly different (P＜0.05).Among HT patients complicated with PTC,tumor multifocality was identified in 32％(26/81) of patients with high level of TSH and in 29％(52/182) of patients with normal level of TSH.The difference had no statistical significance (P＞0.05).Conclusion High preoperative TSH level may stimulate malignant transformation of thyroid tissue in HT patients.