p65基因序列shRNA慢病毒载体构建及其作用功能的初步研究

Objective To obtain shRNA sequences that can stably block the expression of Nuclear Factor kappa- B (p65) in the prostate cancer cell line LNCaP and construct the lentivirus vector.And validate the gene function of p65 in the cell line. Methods According to p65 genetic information, we design siRNA1, siRNA2, siRNA3 those three siRNA sequences targeting the ods area of p65 gene and then form the corresponding four pairs of complementary single strand DNA of shRNA, including the sense strand and the antisense strand. The synthetic shRNA sequence was inserted into the empty pSIH1-H1-copGFP shRNA Vector, and after transfecting the prostate cancer cells , the inhibitory effect of p65 mRNA by different sequences was detected through real-time PCR, and the inhibitory effect of p65 protein expression was detected by Western-blotting. Thus we can obtain highly effective shRNA sequences in the inhibition of p65 in prostate cancer cells. MTT, flow cytometry, transwell were chosen to test the cell growth, migration and invasive power in vitro to compare the difference of the experimental group, control group and negative group. Results The third shRNA sequence had the best inhibitory effect and the inhibitory effect of p65 mRNA in prostate cancer cell line was 59 % and the protein was 81%. It's position locates in p65 (NM_021975 ) 1096-1113 and it's stemloop sequence is 5'-GATCCGCCCTATCCCTTTACGTCATTCAAGAGATGACGTAAAGGGATAGGGCTTTTTG-3'. After transfecting, the prostate cancer cell line had the low expression of p65 stably. Through MTT, we got the growth curve, which showed that the growth ability of experimental group was significantly decreased compared with the control group and the Logarithmic growth didn't appear in the first 96 hours. Flow cytometry test displayed that the percentage of G0-G1-phase cells in experimental group was 61.49%, and the control group was 44.89%, idle group was 41.52%, which was increasing oberviously. The S-phase cells in the experimental group was 28.58%, compared with the 47.36% and 46. 10% diminished. The results of transwell showed that the experimental group had 16. 5000±6. 62076 cells and the other two groups had 45. 6333 13. 54159 and 36. 8333±5. 68412 cells, which showed the invasive power of experimental group was significantly declined(P＜0.05).Conclusions It's successful to obtain shRNA sequences that can stably block the expression of p65 in the prostate cancer cell line LNCaP and construct the lentivirus vector. p65 can positively regulates the biological behavior of prostate cancer LNCaP cell line in the cell growth, migration and invasive power.     

上颌前方牵引联合螺旋扩弓器治疗替牙期骨性Ⅲ类错牙合的效果

的 探讨上颌前方牵引联合螺旋扩弓器治疗替牙期骨性Ⅲ类错牙合的临床效果。方法 选择2021年5月-2022年5月我院收治的70例替牙期骨性Ⅲ类错牙合患儿为研究对象,依据就诊顺序分为对照组和研究组,各35例。对照组给予上颌前方牵引治疗,研究组给予上颌前方牵引联合螺旋扩弓器治疗,比较两组临床疗效、头颅侧位片指标及上气道间隙。结果 研究组治疗总有效率为94.29%,高于对照组的74.27%（P＜0.05）;两组治疗后SNA、SNB、Ptm-A、Y轴角、N-Me比较,差异无统计学意义（P＞0.05）;研究组治疗后ANB、FH-MP、SN-PP、U1-SN、L1-MP、ANS-Me均高于对照组（P＜0.05）;两组治疗后PNS-R均高于治疗前,SPL均低于治疗前（P＜0.05）,但两组治疗后PNS-R、PAS、V-LPW、SPL比较,差异无统计学意义（P＞0.05）。结论 上颌前方牵引联合螺旋扩弓器治疗可有效扩大上气道间隙上部,缩小下部,维持上下颌骨与牙列稳定,疗效确切,在替牙期骨性Ⅲ类错牙合患儿治疗中具有较高的应用价值。     

康复新液预防化疗后消化道反应的疗效

目的 观察恩丹西酮 康复新液预防化疗所致急性、迟发性恶心呕吐的作用及化疗后食欲不振的效果.方法 采用对照方法观察.结果 治疗组控制迟发性恶心呕吐及改善化疗后食欲不振的疗效明显好于对照组.两组对急性恶心、呕吐的有效率无显著性差异.结论 恩丹西酮 康复新液能有效地控制化疗引起的迟发性恶心呕吐,改善化疗后引起的食欲不振.     

高效抑制核因子κ—B的茎环RNA基因序列的获得

目的获得高效抑制前列腺癌细胞株Lncap中核因子kappa—B表达的shRNA序列。方法根据核因子kappa—B基因信息,设计siRNA1、siRNA2、siRNA3三条针对核因子kappa-B基因cds区的siRNA序列及无意义的对照序列,组建与之对应的4对互补的单链DNA序列,包括siRNA的正义链和反义链：正义链序列按5’向3’顺序依次为：酶切位点（BamH Ⅰ）、干扰序列（19bp）、loop环（TFCAAGAGA）、干扰序列的反向互补序列（19bp）、中止信号（TTTTT）、酶切位点（EcoR Ⅰ）。将合成的序列插入空载体pSIH1-H1-copGFP shRNA Vector中,转染前列腺癌细胞后,通过real—timePCR检测不同序列片断对核因子kappa—B的mRNA抑制效果。结果设计的3条针对核因子kappa—B的序列中第3条的抑制效果最好,目的序列位于核因子KAPPA—B（NM_021975）的1096到1113,茎环序列为5＇-GATCC GCCCTATCCCTITACGTCA TTCAAGAGA TGACGTAAAGGGATAGGGC TTITT G-3’。其对前列腺癌细胞株中核因子kappa—B的mRNA的干扰效率为59％,对其蛋白表达的抑制率为81％。转染细胞后,细胞可以稳定低表达核因子kappa—B。结论成功获得高效抑制前列腺癌细胞株Lncap中核因子kappa—B表达的shRNA序列,为后期研究核因子kappa-B在前列腺癌发病中的作用机理等研究提供了基础。     

转基因泡桐树叶及其花粉毒性与过敏性试验

目的初步探讨转入抗菌肽基因的泡桐树叶及花粉的毒性与致敏性。方法采用最大灌胃量法进行泡桐树叶的急性经口毒性试验;按照化妆品卫生规范方法进行花粉的皮肤变态反应试验;采用滴鼻和腹腔注射途径给予豚鼠花粉,观察致过敏性哮喘反应。结果转基因泡桐树（树叶）对SPF级Wistar雌性和雄性大鼠急性经口LD50均〉10000mg/kg·BW;转基因泡桐树（花粉）对普通级白毛豚鼠皮肤变态反应积分为0,致敏率为0%,致敏强度为弱,未见皮肤变态反应;转基因泡桐树花粉对豚鼠未见明显过敏性哮喘反应。结论转入抗菌肽基因泡桐树叶属实际无毒级,花粉对豚鼠皮肤致敏性与过敏性哮喘反应均为阴性。     

腹腔镜、胃镜联合治疗胃穿孔的临床应用

目的:探讨胃镜、腹腔镜联合诊治胃穿孔的可行性及应用价值。方法:回顾分析应用腹腔镜、胃镜联合治疗19例胃穿孔患者的临床资料。术中同时观察胃腔内、腔外病变,修补穿孔。结果:17例手术获得成功,2例术中发现肿瘤或溃疡恶变而中转开腹。手术时间平均100 min,3～5天进流质饮食,术后7～8天出院,无并发症发生。结论:胃镜、腹腔镜联合诊治胃穿孔安全、可行,具有定位准确、患者创伤小、康复快、脏器干扰小等优点,值得临床推广应用。     

王文跃：治愈患者，乃医者创新源动力

王文跃教授说话不温不火．声调浑厚安稳、起伏不大,对患者有抚慰情绪的效果。在他平和的性格之下,则铺垫着诸多“前卫”的思考。多年来,他不断推出很多创新性的探索,而这一切,均源自于他对患者深沉的关爱。     

玉米须中微量元素不同处理方法检测

玉米须是中国传统的中药材料,其所含的微量元素对人体有重要作用,现代药理研究证实,玉米须具有利尿、利胆、降血压、降血糖、抗癌、止血等功效^[1]。     

莫西沙星治疗泌尿系感染80例分析

泌尿系感染是临床常见病、多发病之一，如不及时治疗会引起全身多方面的功能障碍。2005-06-2005-12我院使用莫西沙星治疗泌尿系感染80例，疗效较好，现总结如下。