黏膜佐剂大肠埃希菌不耐热肠毒素B亚单位优化的淋病奈瑟菌孔洞蛋白B核酸疫苗的构建及其诱导小鼠的免疫应答

目的 分析黏膜佐剂大肠埃希菌不耐热肠毒素B亚单位(LTB)辅佐的淋病奈瑟菌孔洞蛋白B(PorB)核酸疫苗诱导小鼠的免疫应答水平.方法 PCR法扩增目的 基因porB、ltB、ltB-porB,分别构建3种相应的peDNA3.1(-)真核重组表达载体,经PCR、双酶切及基因测序鉴定后转染Hela细胞,用细胞免疫荧光法鉴定质粒的蛋白表达.将核酸疫苗经鼻饲免疫雌性BALB/c小鼠,检测体液免疫及细胞免疫应答水平,同时用免疫组织化学法检测porB、ltB、ltB-porB基因在小鼠鼻黏膜内的表达.组间均数比较采用方差分析.结果 构建的真核重组质粒均能在Hela细胞内及小鼠鼻黏膜组织内表达.核酸疫苗免疫组小鼠的生殖道灌洗液PorB特异性sIgA及血清porB特异性IgG水平明显高于对照组(P＜0.01;P＜0.05),且ltB-porB融合基因组特异性sIgA明显高于porB组(P＜0.05);pcDNA3.1(-)/porB组小鼠脾淋巴细胞培养上清液中IFN-γ、IL-4和脾淋巴细胞刺激指数(SI)分别为(170.04±23.89)pg/mL、(114.68±14.27)pg/mL和1.68±0.19,pcDNA3.1(-)/ltB-porB组分别为(161.42±27.50)pg/mL、(124.16±19.04)pg/mL和1.73±0.28,均高于对照组pcDNA3.1(-)和PBS,差异均有统计学意义(P＜0.01;P＜0.05),高于对照组pcDNA3.1(-)/ltB,差异均有统计学意义(均P＜0.05),但两核酸疫苗免疫组间差异无统计学意义(均P＞0.05).结论 所构建的核酸疫苗分别在Hela细胞及小鼠鼻黏膜组织内获得了表达,经黏膜途径免疫能诱导小鼠产生较高水平的特异性体液免疫和细胞免疫应答,尤其是黏膜免疫应答;证实黏膜佐剂LTB可辅佐PorB诱导小鼠产生高水平的生殖道黏膜免疫.

Abstract：

Objective To investigate the specific humoral immune response and cellular immune response induced by DNA vaccine with Neisseria gonorrhoeae porin B (PorB) fused with B subunit of Escherichia coli heat-labile enterotoxin B (LTB) in mice. Methods Target genes of porB, ltB and ltB-porB were amplified by polymerase chain reaction (PCR) and cloned into eukaryotic vector pcDNA3.1(-). The recombinants were identified by PCR, enzyme digestion and DNA sequencing.The vectors were transfected into Hela cells, and expressed proteins were checked by cytoimmunofluorescence. Female BALB/c mice were intranasally immunized with recombination vectors. The humoral immune response and cellular immune response were detected by enzyme linked immunosorbent assay (ELISA) and methyl thiazolyl tetrazolium (MTT) colorimetric assay. The expressions of recombination vectors in intranasal mucosal tissues of the immunized mice were detected by immunohistochemistry. The means between groups were compared by analysis of variance. Results All the three recombinants were expressed in Hela cells and intranasal mucosal tissues. The PorB specific IgG in serum and sIgA in vaginal secretions in DNA vaccine immunized mice were significantly higher than those in controls (P＜0.01 ; P＜0.05). Moreover, the sIgA level in pcDNA3.1 (-)/ltB-porB group was higher than that in peDNA3, 1(-)/porB group (P＝0. 002). The levels of interferon-gamma (IFN-γ) and interleukin-4 (IL-4) in the supernatants and stimulation index (SI) of spleen lymphocyte culture in pcDNA3, 1(-)/porB group were (170.04±23.89) pg/mL, (114.68±14.27) pg/mL and 1. 68±0.19, respectively; and those in pcDNA3, 1(-)/ltB-porB group were (161.42±27.50) pg/mL, (124.16±19.04) pg/mL and 1.73±0.28, respectively; which were both higher than those in pcDNA3.1(-)/ phosphate buffered saliae (PBS) group (P＜0. 01; P＜0.05) and pcDNA3.1 (-)/ltB group (all P＜0.05), while there was no significant difference between pcDNA3.1 (-)/ltB-porB group and pcDNA3. 1 (-)/porB group (0. 998, 0. 696, 0. 994; all P＞0.05). Conclusions The constructed DNA vaccines are all successfully expressed in Hela cells and murine intranasal mucosal tissues. The mucosal immunization of the vaccines [pcDNA3. 1 (- )/porB and pcDNA3.1 ( -)/ltBporB] could induce humoral immune response and cellular immune response, especially mucosal immune response. It is confirmed that mucosal adjuvant LTB could promote PorB to induce higher level of mucosal immune response in mice.     

妈富隆治疗异常子宫出血的临床效果观察

目的探讨妈富隆治疗异常子宫出血(AUB)的临床疗效及其对患者血清卵泡刺激素(FSH)、雌二醇(E_2)、黄体生成素(LH)水平的影响。方法将本院妇科接受治疗的90例AUB患者按照随机数字表法分为对照组与观察组,各为45例,分别给予米非司酮与妈富隆治疗。比较2组临床疗效、止血效果、治疗前后性激素水平、不良反应发生情况。结果观察组临床总有效率为93. 33%,显著高于对照组的71. 11%(P 0. 05);观察组控制出血时间及完全止血时间均显著短于对照组(P 0. 05); 2组患者治疗后FSH、LH及E_2水平均较治疗前显著下降(P 0. 05),且观察组治疗后上述性激素水平降低程度均显著大于对照组(P 0. 05); 2组患者在治疗过程中,均未出现肝、肾功能相关不良反应,仅有个别患者出现轻度恶心、头晕等反应,但对治疗无影响。结论妈富隆可抑制AUB患者血清中FSH的释放水平,促使内源性E_2及LH水平降低,具有很好的性激素调节效果,临床疗效显著、安全性高,可作为临床治疗AUB的首选方案。     

微信联合PBL教学在病理实验教学改革中的研究

目的观察微信联合项目式学习(project-based learning,PBL)教学法在病理实验教学改革中的效果。方法选取本校2016级临床医学专业150名学生作为研究对象,其中3个班级共75名学生设为对照组,该组学生采用传统PBL法进行教学,另外3个班级共75名学生设为观察组,该组学生采用微信联合PBL法进行教学。教学计划完成后,采用笔试考试成绩反映教学效果,采用问卷调查反映学生对教学满意程度,采用大学生自主学习能力评价测评量表评价学生自主学习能力。结果观察组学生笔试考试成绩高于对照组学生[(87.59±7.80)分vs.(82.79±9.79)分],差异有统计学意义(P<0.05)。观察组学生教学满意度评分高于对照组学生[(43.20±3.86)分vs.(38.83±7.00)分],差异有统计学意义(P<0.05)。观察组学生大学生自主学习能力评价测评量表评分高于对照组学生[(91.80±6.95)分vs.(87.08±6.56)分],差异有统计学意义(P<0.05)。结论将微信联合PBL教学法应用于病理实验教学中,可提高学生理论学习成绩,改善学生对教学的满意度,同时对培养学生自主学习能力具有积极的作用。     

研究生实验病理学技术教学改革与实践

病理学技术是实验病理学科研实践的重要专业技能,如何让研究生熟练掌握该项技能是教学中的面临的首要问题.为此,我们通过对研究生实验病理学技能教学过程中存在的问题进行分析,并提出有针对性的教学改革方案.通过近三年的教学实践结果表明,教学改革的实施不仅有利于培养研究生的实验病理学技能,更有利于提高学生的合作意识,创新能力、沟通能力、组织能力等综合素质,可为研究生后续科研实验的顺利开展奠定基础.     

黏膜佐剂大肠埃希菌不耐热肠毒素B亚单位优化的淋病奈瑟菌孔洞蛋白B核酸疫苗的构建及其诱导小鼠的免疫应答

Objective To investigate the specific humoral immune response and cellular immune response induced by DNA vaccine with Neisseria gonorrhoeae porin B (PorB) fused with B subunit of Escherichia coli heat-labile enterotoxin B (LTB) in mice. Methods Target genes of porB, ltB and ltB-porB were amplified by polymerase chain reaction (PCR) and cloned into eukaryotic vector pcDNA3.1(-). The recombinants were identified by PCR, enzyme digestion and DNA sequencing.The vectors were transfected into Hela cells, and expressed proteins were checked by cytoimmunofluorescence. Female BALB/c mice were intranasally immunized with recombination vectors. The humoral immune response and cellular immune response were detected by enzyme linked immunosorbent assay (ELISA) and methyl thiazolyl tetrazolium (MTT) colorimetric assay. The expressions of recombination vectors in intranasal mucosal tissues of the immunized mice were detected by immunohistochemistry. The means between groups were compared by analysis of variance. Results All the three recombinants were expressed in Hela cells and intranasal mucosal tissues. The PorB specific IgG in serum and sIgA in vaginal secretions in DNA vaccine immunized mice were significantly higher than those in controls (P＜0.01 ; P＜0.05). Moreover, the sIgA level in pcDNA3.1 (-)/ltB-porB group was higher than that in peDNA3, 1(-)/porB group (P＝0. 002). The levels of interferon-gamma (IFN-γ) and interleukin-4 (IL-4) in the supernatants and stimulation index (SI) of spleen lymphocyte culture in pcDNA3, 1(-)/porB group were (170.04±23.89) pg/mL, (114.68±14.27) pg/mL and 1. 68±0.19, respectively; and those in pcDNA3, 1(-)/ltB-porB group were (161.42±27.50) pg/mL, (124.16±19.04) pg/mL and 1.73±0.28, respectively; which were both higher than those in pcDNA3.1(-)/ phosphate buffered saliae (PBS) group (P＜0. 01; P＜0.05) and pcDNA3.1 (-)/ltB group (all P＜0.05), while there was no significant difference between pcDNA3.1 (-)/ltB-porB group and pcDNA3. 1 (-)/porB group (0. 998, 0. 696, 0. 994; all P＞0.05). Conclusions The constructed DNA vaccines are all successfully expressed in Hela cells and murine intranasal mucosal tissues. The mucosal immunization of the vaccines [pcDNA3. 1 (- )/porB and pcDNA3.1 ( -)/ltBporB] could induce humoral immune response and cellular immune response, especially mucosal immune response. It is confirmed that mucosal adjuvant LTB could promote PorB to induce higher level of mucosal immune response in mice.     

沙眼衣原体D型生殖道感染小鼠动物模型的建立及评价

目的 建立沙眼衣原体(Chlamydia trachomatis,Ct)D型感染小鼠生殖道动物模型,为研究人类生殖道Ct感染的病理变化和致病机制提供实验基础.方法 分离20个临床株,经阴道接种C3H/HeJ小鼠,得到清除时间明显延长的临床株(UT0603).应用该临床株建立Ct D型感染模型,免疫荧光检测阴道脱落上皮细胞中衣原体的含量;原位组织免疫荧光检测上生殖道的衣原体感染定植情况;分离生殖道组织,肉眼观察并进行病理学评分.结果 Ct 临床株感染小鼠下生殖道其排菌量及排菌周期明显延长,可见衣原体有上行感染和定植,并能引发类似人类生殖道感染的病理变化.结论 成功建立了Ct D型生殖道感染动物模型.     

沙眼衣原体pORF5 蛋白通过激活PI3K / Akt 信号通路抑制细胞凋亡

目的:探讨沙眼衣原体(Chlamydia trachomatis,Ct)pORF5 蛋白对细胞凋亡的影响,并进一步分析其分子机制,为阐明Ct 致病机制提供实验依据。方法:pGEX-6p/ pORF5 重组质粒转化XL1-blue 大肠杆菌,IPTG 诱导表达GST-pORF5融合蛋白,融合蛋白经谷胱甘肽琼脂糖凝胶4B 纯化及蛋白酶切除GST 标签后得到pORF5 蛋白。用不同浓度的pORF5 蛋白刺激HeLa 细胞,Western blot 检测不同时间Bax 和Bcl-2 的表达水平以及PI3K/ Akt 磷酸化水平,Hoechst 33342 及流式细胞技术分析细胞凋亡情况;HeLa 细胞经PI3K/ Akt 特异性抑制剂LY294002 预处理1 h 后,再用pORF5 蛋白刺激24 h,测定细胞凋亡率,并进一步分析凋亡相关蛋白Bax 和Bcl-2 的表达水平及PI3K/ Akt 磷酸化水平。结果:凋亡相关蛋白Bax 和Bcl-2 的表达变化与pORF5 蛋白浓度呈现一定的浓度和时间依赖性,当pORF5 蛋白浓度达10 μg/ ml 时,Bax 表达下调,Bcl-2 的表达上调,当升高至15 μg/ ml 时,Bax 和Bcl-2 的表达量变化最明显;15 μg/ ml 的pORF5 蛋白刺激HeLa 细胞24 h,Bax 和Bcl-2 表达变化最明显;流式细胞检测结果显示:pORF5 蛋白刺激组较TNF鄄琢处理组和未处理组细胞凋亡率分别降低了27.3% (P<0.01)和8.4% (P<0.05);Akt 在pORF5 蛋白刺激15 min 后发生磷酸化,30 min 后磷酸化水平达到峰值,用PI3K 抑制剂LY294002 预处理Hela 细胞后,发现Akt 的磷酸化显著减少,Bax 蛋白表达明显上调,Bcl-2 表达明显下调;LY294002 处理组细胞凋亡率相比于对照组增加了13.0% (P<0.01)。结论:pORF5 蛋白通过激活PI3K/ Akt 信号通路调节Bcl-2 和Bax 蛋白的表达抑制细胞凋亡。     

宫颈横裂皱褶处上皮病理学改变分析

目的：研究宫颈外口横裂皱褶处上皮组织的病理学改变。方法：分析83例Reid阴道镜评分（RCI）为0分的宫颈横裂处上皮的组织病理学结果，并以这83例宫颈的RCI0分非皱褶处上皮组织作为对照组，将两组结果进行比较。结果：83例RCI0分的皱褶处上皮中，22例（26．5％）存在宫颈上皮内瘤变（CIN），其中CINⅠ18例，CIN Ⅱ4例；83例RCI0分的非皱褶处上皮中，6例（7．2％）存在CIN，其中CINⅠ4例，CINⅡ2例。两组比较，差异显著（P〈0．005）。结论：宫颈外口横裂的皱褶处更易发生病变，该处的组织病理学结果常高于阴道镜拟诊，活检时应常规检取皱褶处上皮。     

性交后出血与病理学异常的关系探讨

目的：探讨性交后出血与宫颈癌及其他病理学异常的关系。方法：通过对2000年9月至2002年8月211例以性交后出血为主诉的患者进行宫颈刮片细胞学检查，对其中156例同时行宫颈活体组织病理学检查；并将性交后出血同时刮片异常者73例与同期刮片异常但无性交后出血者进行配对研究。结果：95例宫颈刮片细胞学检查阳性，但明确诊断为宫颈鳞癌或腺癌者14例。经病理学检查证实为宫颈癌者20例，占以性交后出血为主诉者的9．5％，其中有6例宫颈刮片为阴性，假阴性率达30．0％；另有宫颈上皮内瘤样病变(CIN)，炎症反应及宫颈息肉等多种异常。结论：有性交后出血者宫颈癌发生率明显增加，但仍有多种宫颈异常的可能，宫颈涂片细胞学检查阴性并不能排除CIN或浸润癌的可能，应行阴道镜检查及宫颈的活体组织病理学检查。