基于细胞内应变评估的卵母细胞去核方法及在机器人化克隆中的应用

自1996年第一只克隆羊诞生,克隆技术就由于其在动物育种方面的巨大潜力而得到了大量关注。体细胞核移植技术是动物克隆的核心,其操作过程非常复杂,不可避免地造成细胞内损伤。通常来说,只有不到1%的重构胚胎能够发育成克隆动物,克隆的低成功率是制约其广泛应用的主要原因之一。为此,本文提出了一种基于细胞内应变评估的卵母细胞去核方法,来减少体细胞核移植中的潜在细胞内损伤,从而提高克隆成功率。首先,论文根据细胞内速度场计算细胞内应变,进而将内应变作为评价标准来优化去核操作;在此基础上,开发了一套机器人批量体细胞核移植系统,用于实现机器人化克隆。实验结果表明,本文方法将体细胞核移植后的胚胎囊胚率从10.0%提高到20.8%,并成功获得17头克隆小猪,这是世界首批由机器人完成克隆操作获得的克隆动物。对比手动核移植操作,本文将克隆成功率从平均0.73%提升到了2.50%,大幅提升了克隆效率。与此同时,基于细胞内应变的去核方法有望应用于其他生物操作,并建立一种通用的减少细胞内损伤的细胞操作规范。

Inhibitory effect of emodin and Astragalus polysaccharide on the replication of HBV

AIM: To evaluate the anti-viral effect of emodin plus Astragalus polysaccharide (APS) in hepatitis B virus (HBV) transgenic mice.METHODS: Sixty HBV transgenic mice (HBV TGM) whose weight varied between 18 and 24 g were randomly divided into 3 groups, with 20 mice in each group. Group A was the normal control, where the mice were treated with physiological saline; group B was the positive control where the mice were treated with lamivudine solution (100 mL/kg per day). Group C was the experimental group where the mice were treated with physiological saline containing emodin and APS (57.59 mg/kg per day and 287.95 mg/kg per day, respectively). The mice were treated daily for 3 wk. After 1 wk recovery time, the mice were sacrifi ced and serum as well as liver tissues were collected for ELISA and histological examination.RESULTS: After 21 d treatment, HBV DNA levels in group B and group C significantly declined when compared with group A (P < 0.05). However, a signif icant increase in HBV DNA content was observed in group B, whereas this phenomenon was not observed in group C. A reduction in the contents of HBsAg, HBeAg and HBcAg in the mice from group B and C was observed when compared with group A.CONCLUSION: Emodin and APS have a weak but persistent inhibitory effect on HBV replication in vivo, which may function as a supplementary modality in the treatment of hepatitis B infection.

Cytokeratin 8 is increased in hepatitis C virus cells and its ectopic expression induces apoptosis of SMMC7721 cells

AIM:To investigate cytokeratin 8(CK8)overexpression during hepatitis C virus(HCV)infection and its pathogenesis,and the effect of ectopic CK8 expression on hepatoma cell lines.METHODS:We successfully established an in vitro HCV cell culture system(HCVcc)to investigate the different expression profiles of CK8 in Huh-7-HCV and Huh-7.5-HCV cells.The expression of CK8 at the mRNA level was determined by real-time polymerase chain reaction(RT-PCR).The expression of CK8 at the protein level was evaluated by Western blotting.We then constructed a eukaryotic expression combination vector containing the coding sequence of human full length CK8 gene.CK8cDNA was amplified by reverse transcription-PCR and inserted into pEGFP-C1 and the positive clone pEGFPCK8 was obtained.After confirming the sequence,the recombinant plasmid was transfected into SMMC7721cells with lipofectamine2000 and CK8 expression was detected using inverted fluorescence microscopy,RTPCR and Western blotting.Besides,we identified biological function of CK8 on SMMC7721 cells,including cell proliferation,cell cycle and apoptosis detection.RESULTS:RT-PCR showed that the expression level of CK8 in Huh-7-HCV and Huh-7.5-HCV cells was 2.88and 2.95 times higher than in control cells.Western blot showed that CK8 expression in Huh-7-HCV and Huh-7.5-HCV cells was 2.53 and 3.26 times higher than that in control cells,respectively.We found that CK8at mRNA and protein levels were both significantly increased in HCVcc.CK8 was up-regulated in SMMC7721cells.CK8 expression at the mRNA level was significantly upregulated in SMMC7721/pEGFP-CK8 cells.CK8expression in SMMC7721/pEGFP-CK8 cells was 2.69times higher than in SMMC7721 cells,and was 2.64times higher than in SMMC7721/pEGFP-C1 cells.CK8expression at the protein level in SMMC7721/pEGFPCK8 cells was 2.46 times higher than in SMMC7721cells,and was 2.29 times higher than in SMMC7721/pEGFP-C1 cells.Further analysis demonstrated that forced expression of CK8 slowed cell growth and induced apoptosis of SMMC7721 cells.CONCLUSION:CK8 up-regulation might have a functional role in HCV infection and pathogenesis,and could be a promising target for the treatment of HCV infection.

Mechanical effects of needle texture on acupoint tissue

Objective:This study aims to clarify how the stimulation of acupuncture points is achieved by needles with different surface texture during acupuncture;it also seeks to lessen injury at the insertion site and increase the therapeutic efficacy of acupuncture,by simulating the mechanical effects of various needle surface patterns on Zusanli(ST36)without changing the radius of acupuncture needles.Methods:Five acupuncture needle models with different surface patterns,including the smooth needle,the lined needle,the ringed needle,the left-hand threaded needle and the right-hand threaded needle,and a layered model of the Zusanli acupoint were used to investigate how to reduce tissue damage and increase stimulation during acupuncture treatment,Puncturing of the skin as well as liftinginserting and twisting needle manipulations were simulated using these models,and the degree of damage and force of stimulation caused by the acupuncture needles with different surface patterns during acupuncturewerecompared.Results:The smooth needle and the lined needle caused the least tissue damage during insertion,while the left-hand threaded and the right-hand threaded needles caused the most damage.The ringed needle,the left-hand threaded needle and the right-hand threaded needle stimulated the acupoint tissue more during lifting-inserting manipulations,while the lined needle and the smooth needle produced less stimulation.The stimulation of the lined needle on the acupoint tissue was the largest during twisting manipulation,whereas the left-hand threaded needle and the right-hand threaded needle had smaller effects.In lifting-inserting and twisting manipulations,both the left-hand threaded needle and right-hand threaded needle provided more stimulation,but the torsion direction in which they produced better stimulation was theopposite.Conclusion:According to the simulation results,the ringed pattern enhances stimulation best in the lifting-inserting manipulation,whereas the lined pattern enhances stimulation best in the twisting manipulation.Both the right-hand and left-hand thread patterns have certain enhancing effects in these two operations.Taking the geometric properties of the pattern into account,the left-hand thread pattern and the right-hand thread pattern have the geometric characteristics of both the lined pattern and the ringed pattern.To conclude,a pattern perpendicular to the movement direction during the acupuncture manipulation creates more stimulation.These results have significance for future needle design.