中华姬鼠与大林姬鼠的同工酶差异

中华姬鼠（Ａｐｏｄｅｍｕｓｄｒａｃｏ）和大林姬鼠（Ａｐｏｄｅｍｕｓｐｅｎｉｎｓｕｌａｅ）是形态学上十分相似的两种鼠类。为了对两种姬鼠的分类提供生物化学方面的依据,采用聚丙烯酰胺凝胶等电聚焦电泳方法比较和分析了两种姬鼠的ＬＤＨ同工酶、ＥＳＴ同工酶和ＳＯＤ同工酶的差异。结果表明,两种姬鼠的ＬＤＨ同工酶酶谱基本相似,而ＥＳＴ同工酶和ＳＯＤ同工酶酶谱则存在明显的种间差异。根据ＥＳＴ同工酶Ａ２带的有无和ＳＯＤ同工酶主带等电点的差别,能将两种姬鼠很容易区分开来。     

一种简单、经济的植物RNA抽提方法

一种简单、经济的植物ＲＮＡ抽提方法孟玲谭德勇王焕效吕朝晖王晓燕周翔（云南大学生物系,昆明６５００９１）ＡＳｉｍｐｌｅＥｃｏｎｏｍｉｃａｌＭｅｔｈｏｄｆｏｒｔｈｅＩｓｏｌａｔｉｏｎｏｆＰｌａｎｔＲＮＡＭＥＮＧＬｉｎｇＴＡＮＤｅｙｏｎｇＷＡＮＧＨｕａｎｘ...     

Environmental controls over carbon exchange of three forest ecosystems in eastern China

Net ecosystem productivity (NEP) was continuously measured using the eddy covariance (EC) technique from 2003 to 2005 at three forest sites of ChinaFLUX. The forests include Changbaishan temperate mixed forest (CBS), Qianyanzhou subtropical coniferous plantation (QYZ), and Dinghushan subtropical evergreen broad‐leaved forest (DHS). They span wide ranges of temperature and precipitation and are influenced by the eastern Asian monsoon climate to varying extent. In this study, we estimated ecosystem respiration (RE) and gross ecosystem productivity (GEP). Comparison of ecosystem carbon exchange among the three forests shows that RE was mainly determined by temperature, with the forest at CBS exhibiting the highest temperature sensitivity among the three ecosystems. The RE was highly dependent on GEP across the three forests, and the ratio of RE to GEP decreased along the North–South Transect of Eastern China (NSTEC) (i.e. from the CBS to the DHS), with an average of 0.77 ± 0.06. Daily GEP was mainly influenced by temperature at CBS, whereas photosynthetic photon flux density was the dominant factor affecting the daily GEP at both QYZ and DHS. Temperature mainly determined the pattern of the interannual variations of ecosystem carbon exchange at CBS. However, water availability primarily controlled the interannual variations of ecosystem carbon exchange at QYZ. At DHS, NEP attained the highest values at the beginning of the dry seasons (autumn) rather than the rainy seasons (summer), probably because insufficient radiation and frequent fog during the rainy seasons hindered canopy photosynthesis. All the three forest ecosystems acted as a carbon sink from 2003 to 2005. The annual average values of NEP at CBS, QYZ, and DHS were 259 ± 19, 354 ± 34, and 434 ± 66 g C m⁻² yr⁻¹, respectively. The slope of NEP that decreased with increasing latitude along the NSTEC was markedly different from that observed on the forest transect in the European continent. Long‐term flux measurements over more forest ecosystems along the NSTEC will further help verify such a difference between the European forest transect and the NSTEC and provide insights into the responses of ecosystem carbon exchange to climate change in China.     

HBV pre-c-Fc融合蛋白在杆状病毒表达系统中的表达及其生物学活性研究

目的:在杆状病毒表达系统中表达融合蛋白乙型肝炎病毒前C蛋白-小鼠IgG Fc蛋白(HBV precore protein-mouse IgG Fc,HBV pre-c-Fc),并鉴定其免疫原性。方法:目的基因HBV prec-Fc连接到pFastBac1载体,获得的pFastBac1-HBV pre-c-Fc质粒转化DH10Bac感受态,通过Tn7转座子将目的基因转座到Bacmid中,得到Bacmid-HBV pre-c-Fc穿梭载体,脂质体包被后转染Sf9昆虫细胞获得P1代病毒,重复转染Sf9获得高滴度病毒。收集细胞上清超滤后通过Protein G亲和层析柱纯化得到目的蛋白HBV pre-c-Fc。纯化的蛋白大腿内侧肌肉注射免疫BALB/c小鼠并检测血清中乙型肝炎病毒核心蛋白抗体产生量。结果:HBV pre-c-Fc在昆虫细胞中成功表达,纯化后蛋白纯度达90%以上,蛋白产量约为3.03mg/L,纯化蛋白能有效刺激BALB/c小鼠产生特异抗体。结论:成功地在杆状病毒表达系统中表达了具有免疫原性的HBV pre-c-Fc蛋白,为生产乙肝治疗性疫苗奠定了基础。     

在大肠杆菌中表达酮酸脱羧酶产异戊醇

酮酸脱羧酶作为异戊醇生物合成的关键酶,不存在于大肠杆菌中。以乳酸乳球菌的基因组DNA为模板,经过PCR扩增得到酮酸脱羧酶基因kivD(rbs),插入到大肠杆菌高效表达载体pET-28a(+)上形成pET-kivD(rbs),重组质粒热击转化进大肠杆菌BL21(DE3)中,其成功表达了酮酸脱羧酶。对发酵产物进行分析,检测到了微量的目标产物—异戊醇。     

人源天然ScFv噬菌体抗体库的构建及鉴定

噬菌体抗体库技术是获得治疗性抗体的一条重要途径。以20份健康人外周血为样本,通过提取淋巴细胞、逆转录－PCR（RT PCR）、抗体可变区基因的扩增、重叠PCR获得单链抗体（ScFv）基因,将ScFv克隆入噬粒载体,通过近300次的电转化获得了库容量为1.3×109的全人源天然ScFv噬菌体抗体库。通过随机挑克隆测序和用5种不同抗原筛选对抗体库进行了初步验证。随机测序表明抗体库具有较好的多样性,用5种不同抗原对其进行筛选,均获得了特异性噬菌体抗体的不同富集,表明成功构建了一个多样性良好的人源天然ScFv噬菌体抗体库。     

CpG island hypermethylation of multiple tumor suppressor genes associated with loss of their protein expression during rat lung carcinogenesis induced by 3-methylcholanthrene and diethylnitrosamine

The epigenetic mechanisms underlying the tumorigenesis caused by polycyclic aromatic hydrocarbons and nitrosamine compounds such as 3-methylcholanthrene (MCA) and diethylnitrosamine (DEN) are currently unknown. We reported previously that dynamic changes in DNA methylation occurred during MCA/DEN-induced rat lung carcinogenesis. Here, we used the same animal model to further study the evolution of methylation alterations in tumor suppressor genes (TSGs) DAPK1, FHIT, RASSF1A, and SOCS-3. We found that none of these genes were methylated in either normal or hyperplasia tissue. However, as the severity of the cancer progressed through squamous metaplasia and dysplasia to carcinoma in situ (CIS) and infiltrating carcinoma, so methylation became more prevalent. Particularly dramatic increases in the level of methylation, the average number of methylated genes, and the incidence of concurrent methylation in three genes were observed in CIS and infiltrating carcinoma. Similar but less profound changes were seen in squamous metaplasia and dysplasia. Furthermore, methylation status was closely correlated to loss of protein expression for these genes, with protein levels markedly declining along the continuum of carcinogenesis. These results suggest that progressive CpG island hypermethylation leading to inactivation of TSGs might be a vital molecular mechanism in the pathogenesis of MCA/DEN-induced multistep rat lung carcinogenesis.     

冠心病患者血清CRP的变化及其临床意义

目的:探讨冠心病(CAD)患者血清C-反应蛋白(CRP)的变化及其临床意义.方法:运用全自动生化分析仪检测97例CAD患者和28例对照组人群的血清CRP,将CAD患者进一步分为稳定性心绞痛组(SAP)、不稳定心绞痛(UAP)、心肌梗死组(AMI)三组.另外,将97例CAD惠者分为1支病变组、2支病变组和3支病变组.所有研究对象均行选择性冠状动脉造影检查.结果:(1)与对照组比较,CAD患者血清CRP显著升高(P<0.05);(2)与对照组比较,SAP组、UAP组和AMI组患者血清CRP均显著升高(P<0.05);与SAP组比较,UAP组和AMI组患者血清CRP亦显著升高(P<0.05);(3)与对照组比较,1支病变组、2支病变组和3支病变组患者血清CRP均显著升高(P<0.05);与1支病变组比较,2支病变组和3支病变组患者血清CRP亦显著升高(P<0.05);与2支病变组比较,3支病变组患者血清CRP亦显著升高(P<0.05).结论:(1)CRP可能与CAD有关;(2)CRP异常升高可能反映冠状动脉斑块的不稳定和CAD的严重程度.     

大鼠脑缺血再灌注后Cathepsin D和caspase-9表达的动态变化

目的:探讨大鼠缺血再灌注后溶酶体组织蛋白酶D(Cathepsin D CD)、caspase-9在不同时段蛋白质及mRNA表达变化。方法:将60只S D大鼠随机分为三组:正常组(10只),假手术组(10只),脑缺血再灌注组(40只),线栓法制备大脑中动脉梗死模型(MCAO),免疫组化及RT-PCR法分别检测Cathepsin D、caspase-9的蛋白和mRNA表达。结果:与正常组和假手术组比较,模型组大鼠脑缺血再灌注损伤后6h Cathepsin D的蛋白和mRNA表达明显增强(P<0.05),24h达高峰,48h仍保存高水平。caspase-9蛋白和mRNA6h开始明显升高,12h达高峰,此后缓慢下降,但48h组仍显著高于对照组(P<0.05),结论:Cathepsin D、caspase-9在大鼠脑缺血再灌注后表达增强,溶酶体可能参与了脑缺血再灌注损伤后神经细胞凋亡的过程。     

COX-2在CIN及宫颈癌组织中表达的意义

目的:探讨前列腺素酶COX-2在宫颈癌及癌前组织中的表达及意义.方法:采用免疫组化SP法、半定量RT-PCR法及Western-blot法检测COX-2在35例宫颈上皮肉瘤样病变(CIN)、40例宫颈癌和5例正常宫颈组织中的表达情况.结果:①经免疫组化检测,COX-2蛋白在正常宫颈组织中不表达,在CIN宫颈组织阳性表达率分别为31.4%,在宫颈癌组织中阳性表达率分别为85.0%.三者之间差异具有显著性(P<0.05).②经RT-PCR检测,正常宫颈组织中无COX-2 mRNA表达,CIN宫颈组织中COX-2mRNA相对含量为0.291±0.214,宫颈癌组织中相对含量为0.357±0.173,三者之间差异具有显著性(P<0.05).③经Western-blot检测.COX-2蛋白在宫颈癌Ⅰ、Ⅱ、Ⅲ期中相对含量分别为40.27±13.67,56.98±21.13和84.21±22.74,三者之间差异具有统计学意义(P<0.05).结论:COX-2蛋白不表达于正常宫颈组织中.COX-2可能参与了宫颈癌早期的发生发展.宫颈组织中COX-2的表达可作为宫颈癌早期诊断及估计宫颈癌患者预后的一个指标.