TIMP-2逆转录病毒载体的构建及表达

目的构建人组织型基质金属蛋白酶抑制剂2（TIMP-2）编码序列基因逆转录病毒表达载体，转染人单核细胞白血病细胞株，为探讨TIMP-2的生物学功能奠定基础。方法根据基因库中已公布的序列设计引物，用PCR方法扩增出人TIMP-2编码序列基因片段，用限制性内切酶及T4连接酶将目的片段插入MSCV逆转录病毒载体中。采用酶切和PCR鉴定及测序后经脂质体介导转染至PT67包装细胞中，以病毒上清感染单核细胞白血病细胞株SHI-1，G418筛选，极限稀释法挑选单克隆细胞株，定量PCR和Western Blotting检测TIMP-2的表达。结果TIMP-2基因克隆进逆转录病毒载体MSCV中，经酶切、PCR和DNA测序证实重组逆转录病毒载体构建正确，病毒上清感染SHI-1细胞，经G418筛选并挑选出单克隆细胞，经实时定量PCR和Western Blotting检测发现SHI-1细胞中TIMP-2 mRNA及蛋白表达明显上调。结论成功构建了高表达TIMP-2的SHI-1细胞，可对其生物学行为作进一步研究。     

胰岛素基因逆转录病毒载体在人骨髓间充质干细胞表达的实验研究

目的构建携带胰岛素基因逆转录病毒载体,使其在人骨髓间充质干细胞(hMSCs)内稳定表达。方法通过RT-PCR法从人胰腺组织中提取胰岛素基因片段,并将其连接入逆转录病载体pLNCX,经包装细胞PT67的包装后,获得含外源性胰岛素基因的病毒颗粒(病毒滴度达4.2×106cfu/ml)并感染hMSCs,经过G418筛选后,用RT-PCR法、免疫荧光法、放射免疫法、葡萄糖刺激试验分析胰岛基因的表达情况。结果携带胰岛素基因的逆转录病毒载体构建成功,转导入hMSCs后能够稳定表达胰岛素基因,并分泌至细胞外持续3周以上,但对葡萄糖刺激无明显反应。结论重组人胰岛素基因逆转病毒载体能够将胰岛素基因导入hMSCs并稳定表达。     

绿色荧光蛋白和酪氨酸羟化酶在骨髓基质细胞中的表达

目的研究反转录病毒介导的绿色荧光蛋白(EGFP)基因和酪氨酸羟化酶(TH)基因在原代培养的骨髓基质细胞(bMSCs)中的表达情况。方法体外分离培养大鼠四肢骨的bMSCs,原代培养8~10 d后,同时利用含EGFP和TH基因的反转录病毒上清依次转染bMSCs并进行抗性筛选,7~10 d可得到稳定表达EGFP的bMSCs,在稳定转染EGFP后10 d进行TH病毒上清的转染,7~10 d可得到稳定表达TH和EGFP的bMSCs。结果原代培养bMSCs 3~4 d后生长至70%~80%汇合,病毒转染EGFP后第2天,细胞呈现弱荧光,经G418筛选2~3 d后出现阳性细胞,约8~10 d生长融合,阳性率60%~70%,第2~5代阳性细胞比率无明显变化。TH阳性率也达60%以上。结论利用反转录病毒载体可有效地将EGFP和TH基因转至bMSCs中,并能在体外稳定表达传代。这种表达EGFP和TH的bMSCs对于研究bMSCs的体内迁移分化及帕金森病(PD)的基因治疗具有重要意义。     

表达载体sHLA-A2-IgG1-Fc的构建与鉴定

目的构建表达载体pcDNA3.0-sHLA-A2-IgGl-Fc,为进一步真核表达可溶性HLA-A2-IgG1-Fc蛋白奠定基础。方法从T2细胞中提取总RNA,借助RT-PCR技术扩增包括信号肽的可溶性HLA-A2的cDNA序列并把其插入真核表达载体pcDNA3.0,然后经酶切和测序法鉴定;用PCR的方法从含IgG1-Fc基因的PIG质粒中扩增目的基因IgG1-Fc段,然后经酶切后插入前面构建好的表达载体pcDNA3.0-sHLA-A2中,最后经酶切和测序法鉴定。结果经酶切鉴定及测序分析,证实已将目的基因HLA-A2和IgG1-Fc片段插入载体pcDNA3.0。结论本研究成功构建表达载体pcDNA3.0-sHLA-A2-IgG1-Fc。     

tRNA~(val)-CTE复合核酶真核表达载体构建

目的构建tRNA^val—CTE复合核酶真核表达载体，为进一步研究打下基础。方法用RT—PCR法扩增CTE DNA；合成含tRNA^val启动子和HCV5-NCIK区195住的核酶以覆内切酶KpnⅠ和EcoRⅤ序列，通过退火连接到pPUR质粒的BamHⅠ和EcoRⅠ的酶切位点之间，构建质粒pPHCV—Rz；用KpnⅠ和EcoRⅤ双酶切后，将CTE cDNA定向插入表达载体pPHCV—Rz多克隆位点中，构建成重组表达质粒，并用酶切分析和序列测定进行了鉴定。结果酶切鉴定和测序证实tRNA^val启动子和HCV5-NCIK区195住的核酶基因以覆CTE cDNA被正确克隆入真核表达载体pPUK。结论tRNA^val-CTE复合核酶真核表达载体的成功构建，为开展利用tRNA^val-CTE复合核酶切割HCV RNA的研究奠定了基础。     

AIF重组质粒的构建、表达和功能的研究

目的构建人凋亡诱导因子(AIF)基因功能片段的真核表达载体pcDNA3.1( )AIF,并初步研究AIF在细胞凋亡中的作用。方法从人细胞中提取总RNA,RTPCR扩增AIF基因片段;克隆到pMD18T载体上,通过酶切和测序进行鉴定;将AIF基因片段插入pcDNA3.1( ),构建真核表达载体;电转化淋巴母细胞Raji细胞,观察AIF的表达水平和功能。结果成熟AIF蛋白分子在真核细胞中表达,并转移到细胞核中,诱导Caspase非依赖性细胞凋亡。结论成功构建了pcDNA3.1( )AIF真核表达载体,AIF在Caspase非依赖性细胞凋亡中发挥重要作用。     

Proprioceptive neck influences modify the information about tilt direction coded by the cerebellar anterior vermis

Objective To verify whether the direction of head tilt coded by the population response of Purkinje (P) cells located in the cerebellar anterior vermis is modified by the relative position of the body with respect to the head.

Material and Methods In decerebrate cats, the responses of P cells to wobble of the whole animal were analyzed in order to compute the response vectors of the same cells to the labyrinthine input. These response vectors were used to evaluate the population response (vector) of all the recorded neurons to head tilt in specific directions.

Results When the head was aligned with the body, the direction of the population vector closely corresponded to that of head tilt. A 30° body-to-head rotation to the left or right around a C1–C2 vertical axis modified the response vectors of most of the recorded neurons, leading to reciprocal deviations of the population vector from the direction of head tilt of ≈30°.

Conclusion We propose that information from neck receptors regulates the convergence of labyrinthine signals with different spatial and temporal properties on corticocerebellar units, thus allowing the P-cell population to code the direction of body tilt.     

One-year persistence of individual gait patterns identified in a follow-up study – A call for individualised diagnose and therapy

Although a hunch about the individuality of human movements generally exists, differences in gait patterns between individuals are often neglected. To date, only a few studies distinguished individual gait patterns in terms of uniqueness and emphasised the relevance of individualised diagnoses and therapy. However, small sample sizes have been a limitation on identifying subjects based on gait patterns, and little is known about the permanence of subject-specific characteristics over time. The purpose of this study was (1) to prove the uniqueness of individual gait patterns within a larger sample and (2) to prove the long-term permanence of individual gait patterns. A sample of 128 healthy participants each walked a distance of 10 m barefoot 10 times. Two force plates recorded the ground reaction forces during a double step at a self-selected walking speed. A subsample of 46 participants repeated this procedure after a period of 7–16 months. The application of support vector machines resulted in classification rates of 99.8% (1278 out of 1280) and 99.4% (914 out of 920) for the initial subject-classification and the subsample follow-up-classification, respectively. The results showed that gait patterns based on time-continuous ground reaction forces were unique to an individual and could be differentiated from those of other individuals. Support vector machines classified gait patterns to the corresponding individual almost error-free. Hence, human gait is not only different between individuals but also exhibits unique individual characteristics that are persistent over years. Our findings provide evidence for the individual nature of human walking and emphasise the need to evaluate individualised clinical approaches for diagnoses and therapy.