梅花鹿ACP1基因cDNA克隆及序列分析

为了初步探索梅花鹿ACP1基因的功能及其在鹿茸发育和生长过程中的作用,试验采用RT-PCR技术和分子克隆技术等获得了梅花鹿ACP1基因cDNA序列,并利用软件对获得的梅花鹿ACP1基因cDNA序列进行生物信息学分析。结果表明:cDNA序列长758 bp,CDS长477 bp,编码158个氨基酸,相对分子质量为18 026.57;整个氨基酸组成中,丙氨酸(Ala)和天冬氨酸(Asp)所占比例最高,达到7.6%,理论等电点(pI)为7.51,不稳定系数为41.71,是不稳定蛋白;ACP1蛋白为亲水性蛋白,有信号肽,位点为25~26:AEA^VF;ACP1蛋白的二级结构为无规则卷曲62.03%,延伸链18.99%,α-螺旋18.99%;梅花鹿的ACP1基因与家牛、野骆驼亲缘关系较近。

体外RNA干扰抑制ACP1对骨肉瘤细胞侵袭的影响

目的探讨ACP1蛋白对人骨肉瘤细胞侵袭能力的影响。方法以骨肉瘤细胞MG-63为研究对象,针对ACP1基因设计小干扰RNA(si RNA),构建其短发夹状RNA(short hairpin RNA,shRNA)真核表达载体并转染入细胞,在mRNA和蛋白水平检测ACP1表达,通过黏附实验、细胞划痕、transwell检测MG-63体外侵袭能力。结果成功构建si RNA表达载体Pgenesil-1/ACP1-shRNA。shRNA可使ACP1在基因和蛋白表达水平显著降低,骨肉瘤细胞的迁移能力明显下降,黏附能力由(96.4±8.8)%到(43.4±6.0)%,P<0.01;穿过人工基底膜的细胞数量明显减少,由56.5±4.8减少到36.3±6.1,P<0.05。结论ACP1在骨肉瘤的侵袭过程中发挥着重要作用,为肿瘤的生物学治疗提供了新思路。

IGIACP1 predicts the prognosis in multiple myeloma patients

Objective The aim of this study was to investigate the prognostic relevance of acid phosphatase 1(ACP1) expression in myeloma patients by using Gene Expression Omnibus(GEO) datasets.Methods A comprehensive search was performed in the GEO database in order to find appropriate datasets. The expression level of ACP1 was extracted from the dataset involving both newly diagnosed and relapsed myeloma patients, and a comparison was made. Clinical follow-up data and ACP1 expression were extracted, and survival analysis of overall survival was performed to compare the high-(top quartile) and low-expression(bottom quartile) groups. Analyses using Kaplan-Meier estimation, log-rank test, and restricted mean survival time(RMST) comparison were performed.Results The GSE 6477 dataset was used to make a comparison of the ACP1 expression levels among patients with newly diagnosed and relapsed myeloma. The ACP1 expression level was significantly higher in the relapsed group than in the newly diagnosed group [mean difference =-262.9, 95% confidence interval(CI) =(-420.2,-105.5), P = 0.002]. The GSE 2658 dataset was used for investigating the prognostic relevance of ACP1 expression in myeloma. The ACP1 high-expression group had a significantly worse prognosis [low vs high: hazard ratio = 0.54, 95% CI =(0.31, 0.95); χ2 = 5.02, log rank P = 0.0314]. The median survival was 55.9 months in the high-expression group and was not reached in the low-expression group. The restricted mean time loss(95% CI) was 11.03(12.97, 23.11) and 18.04(12.97, 23.11) for the low-and high-expression groups, respectively. The ratio of RMST(95% CI) between the two groups(high vs low) was 0.87(0.77, 0.99; P = 0.03).Conclusion Our study, for the first time, showed that ACP1 predicts the prognosis in multiple myeloma patients. Further studies are needed to determine the potential mechanism by which ACP1 is associated with clinical outcomes and should focus on the differential roles of low-molecular-weight protein tyrosine phosphatase(LMWPTP) isoforms.

The influence of down-regulation of ACP1 by RNAi on the metastasis capability of osteosarcoma cell line MG-63

Objective： The aim of this study was to study the inhibition effect of small interfering RNAs （siRNA） on gene expression in MG-63 cells,and to study the inhibitory effect on metastasis of MG-63. Methods： A plasmid of a short hairpin RNA targeting acid phosphatase 1 （ACP1） was constructed and transfected into MG-63 cell line. ACP1 expression of MG-63 cells before and after transfection was detected by RT-PCR and Western blot. The capacity of adhesion, migration and inva- sion was examined by adhesion assay, migration assay and transwell assay. Results： The recombinant plasmid pGenesil-l/ ACPI-shRNA was successfully constructed, shRNA efficiently inhibited the expression of ACPI by gene and protein level and suppressed cell migration. The adhesion decreased from 96.41±8.83 to 43.38 ± 6.03 （P 〈 0.01）, invasion ability from 56.5 ± 4.8 to 36.3 ± 6.1 （P 〈 0.05）. Conclusion： Down-regulating ACP1 by shRNA reduced the capacity of metastasis of MG-63 cell,which providing a novo-approach to biotherapy of cancer.

天冬多糖对小鼠免疫功能影响的实验研究

目的:探讨天冬多糖对小鼠免疫功能的影响。方法:通过小鼠胸腺和脾脏指数的测定,确定了小鼠巨噬细胞吞噬的功能、淋巴细胞转化率和血清溶血素的形成。结果:天冬多糖的高、中、低剂量明显能使小鼠腹腔巨噬细胞吞噬鸡红细胞百分率、吞噬指数和胸腺、脾脏等免疫器官重量的增加,从而使小鼠淋巴细胞转化率及胸腺、脾脏指数的增加和小鼠血清溶血素水平的提高。结论:天冬多糖有增强机体免疫功能的作用。

基于质粒分子的转cry1Aa基因棉花定量PCR方法的建立

【目的】转cry1Aa基因棉花南农6号为我国未经批准商业化种植的棉花品种。为监测其非法种植,解决阳性标准品不容易获得的问题,构建适合转cry1Aa基因棉花南农6号品系特异性检测的质粒分子pMD-NN6,以该质粒分子作为标准物质,建立相应的实时荧光定量PCR方法。【方法】根据南农6号转化体特异序列和棉花内标准基因acp1设计引物,采用重叠PCR方法构建质粒分子;以质粒分子作为标准物质,南农6号转化体特异序列为靶标,建立了南农6号棉花特异性定量检测方法。【结果】构建的质粒全长为3148 bp,含有南农6号品系特异性序列和棉花acp1基因序列两个靶标片段的质粒分子,定量标准曲线斜率和扩增效率均符合要求,定量检测限为30拷贝。两个已知南农6号含量的混合样品(2.0%和0.5%)定量检测的准确度标准偏差均在±25%范围内,精确度相对标准差均≤25%。【结论】建立的PCR定量检测方法可以用于含有南农6号转基因棉花产品的品系特异性定量检测,所构建的标准质粒pMD-NN6可以代替其基因组DNA作为标准物质使用。

Smoking, ACP₁and Infertility in Subjects with Varicocele

Several studies suggest that smoking may have detrimental effect on seminal parameters and that genetic polymorphisms may influence this effect. In the present paper we have examined the relationship between ACP1 genotypes and the association of smoking and infertility in a sample of men with varicocele. 126 subjects with varicocele were studied. ACP1 genotype was determined by DNA analysis and statistical analyses were performed by SPSS software. In general there is a protective action of low activity *A/*B genotype against infertility that is much more marked in smokers than in non-smokers. If confirmed in other clinical settings, determinations of ACP1 genotype could have practical importance to evaluate the risk of infertility in smokers with varicocele.

白念珠菌酰基载体蛋白1的制备

目的 :通过在大肠埃希菌BL21中表达白念珠菌酰基载体蛋白1(acyl carrier protein 1,Acp1),以制备高纯度的目的蛋白。方法:采用PCR扩增目的基因ACP1后,在序列的C端拼接上6xHis tag及终止子,拼接完成后连接构建到质粒中,成为原核表达载体pET30a-ACP1,转化入大肠埃希菌感受态细胞BL21中,通过异丙基-β-D-硫代半乳糖苷(isopropyl-beta-D-thiogalactopyranoside, IPTG)诱导表达。收集表达产物后通过镍离子螯合柱纯化,最终利用考马斯亮蓝Bradford法测得目的蛋白含量。结果:白念珠菌酰基载体蛋白Acp1在大肠埃希菌BL21中高效表达;抽提纯化蛋白后行蛋白电泳检测,结果显示,在还原条件下Acp1一直存在2条相对分子质量为17 000的条带。经还原和非还原电泳及蛋白印迹法检测,结果显示该蛋白在非还原状态下有聚集。利用Bradford方法测得蛋白Acp1浓度为2.68 mg/mL。结论:成功制备了白念珠菌酰基载体蛋白Acp1,为后期利用荧光偏振法筛选靶点为Ppt2酶的临床药物提供基础。