目的 利用大肠杆菌原核表达系统优化表达纯化肠道病毒71型(EV71)VP3结构蛋白,为后续单克隆抗体制备及检测试剂盒研发提供前期基础。方法 采用PCR方法扩增EV71病毒VP3基因,将其插入表达载体pET28a(+),构建pET28a-VP3重组质粒,转化大肠杆菌BL21(DE3)菌株,分别在25 ℃、37 ℃下经IPTG诱导表达,重组表达的蛋白产物经凝胶电泳初步分析,比较不同温度诱导表达的蛋白产物。结果 成功构建pET28a-VP3重组质粒,不同温度下诱导表达的蛋白产物在30.5 kDa左右位置均出现目的条带;37 ℃下诱导表达的蛋白超声破碎并离心后,目的蛋白基本位于沉淀中,而25 ℃诱导表达的蛋白产物有少量目的蛋白溶解于上清液中。结论 在25 ℃或37 ℃下均能利用大肠杆菌原核表达系统有效表达EV71病毒VP3蛋白;37 ℃诱导时蛋白可融性表达低,目的蛋白获取效率较高。

Objective To express VP3 capsid protein of enterovirus 71 by using Escherichia coli prokaryotic expression system. Methods VP3 gene was amplified by PCR before inserted into pET28a(+) plasmid. Then the plasmid pET28a-VP3 was transformed and expressed in the Escherichia coli BL21 strain at 25 ℃ or 37 ℃. Finally the protein was analyzed by SDS-PAGE gel electrophoresis. Results The pET28a-VP3 plasmid was successfully constructed, and the EV71 VP3 protein was expressed. Supernatant of the production after ultrasonication and centrifugation got a little VP3 protein. Conclusion The EV71 VP3 protein was expressed. Expression at 25 ℃ may lead to the dissolution of the recombinant protein.

目的 观察芳香烃受体(AhR)及Th17相关细胞因子在类风湿关节炎中的表达水平及其对疾病的预测价值。方法 选择2014年1月—2015年12月于我院就诊的RA患者60例作为观察组,选取同期于我院进行健康体检的正常人60例作为对照组,采用酶联免疫吸附法检测血清中 IL-17、IL-23及AhR的表达水平,并分析其与疾病活动度的关系。结果 RA患者血清IL-17、IL-23及AhR水平高于对照组(P＜0.05)。而根据病情严重程度,RA 非早期组IL-17、IL-23及AhR水平较早期组增高(P＜0.05)。血清 IL-17 水平与除CRP以外的病情活动度指标均呈正相关关系(P＜0.05)。IL-23 水平与SJC、TJC、HAQ 、DAS28 评分呈正相关关系(P＜0.05),但其他指标无明显相关性(P>0.05)。RA患者PBMC中AhR的表达水平与各项临床指标无相关性(P>0.05)。IL-17和IL-23水平与Sharp评分呈正相关关系(r=0.895,P＜0.01;r=0.708,P＜0.01)。AhR的表达与血清IL-17和IL-23水平呈正相关(r=0.415,P＜0.01)。经荧光定量PCR检测结果显示,RA患者AhR及其下游因子CYP1A1、IL-17、FOXP3 mRNA的表达水平高于对照组(P＜0.05)。且RA 非早期组AhR及其下游因子CYP1A1、IL-17、FOXP3 mRNA的表达水平较早期组增高(P＜0.05)。经相关关系检测,RA患者AhR及其下游因子CYP1A1、IL-17、FOXP3 mRNA的表达水平与Sharp评分呈正相关关系(r=0.715,P＜0.01;r=0.734,P＜0.01;r=0.812,P＜0.01;r=0.755,P＜0.01)。结论 Th17相关细胞因子IL-17和IL-23在RA病理生理过程中发挥重要作用,其表达增高,提示关节炎症处于活跃状态,骨质破坏较重,可作为评估RA病情的重要指标。RA患者体内AhR蛋白及其相关下游信号通路均呈高表达状态,AhR通路在RA患者的发病过程中可能发挥关键作用。

Objective To observe the expression level of aroma receptor (AhR) and Th17 related cytokines in rheumatoid arthritis and its predictive value to disease. Methods Sixty patients with RA who were treated in our hospital from January 2014 to December 2015 were selected as the observation group. Sixty healthy subjects were randomly selected as the control group. IL-17, IL-23 and AhR levels in serum were detected by enzyme-linked immunosorbent and the relationship between IL-17, IL-23 and disease activity were analyzed. Results IL-17, IL-23 and AhR levels in serum of RA patients were significantly higher than those in controls (P<0.05). However, the levels of IL-17, IL-23 and AhR levels were significantly higher in the non- early RA group than in the early group (P<0.05), depending on the severity of the disease. There was a positive correlation between serum IL-17 level and disease activity index except CRP (P<0.05). IL-23 level was positively correlated with SJC, TJC, HAQ and DAS28 scores (P<0.05), but no significant correlation was found between other indexes (P>0.05). The expression level of AhR in PBMC of RA patients was not correlated with clinical indexes (P>0.05). IL-17 and IL-23 levels were positively correlated with Sharp score (r=0.895, P<0.01; r=0.708, P<0.01). The expression of AhR was positively correlated with serum IL-17 and IL-23 levels (r=0.415, P<0.01). The expression of AhR and its downstream factors CYP1A1, IL-17 and FOXP3 mRNA in RA patients were significantly higher than those in the control group (P<0.05) by fluorescence quantitative PCR. The expression of AhR and its downstream factors CYP1A1, IL-17 and FOXP3 mRNA in RA group were significantly higher than those in early group (P<0.05). The expression level of AhR and its downstream factors CYP1A1, IL-17 and FOXP3 mRNA in RA patients were positively correlated with Sharp scores (r=0.715, P<0.01; r=0.734, P<0.01; r=0.812, P<0.01; r=0.755, P<0.01). Conclusion The Th17-related cytokines IL-17 and IL-23 play an important role in the pathophysiology of RA, and the expression of Th17-associated cytokines is increased, suggesting that arthritis is active and bone destruction is serious. The AhR protein and its associated downstream signaling pathways are highly expressed in RA patients, and the AhR pathway may play a key role in the pathogenesis of RA patients.

目的 观察乳腺癌组织中沉默信息调节因子(SIRT1)的表达并探讨其临床意义。方法 选取70例乳腺癌组织和20例乳腺纤维腺瘤组织,应用免疫组化法观察SIRI1在两组中的表达,并分析其与临床病理参数、预后之间的关系。结果 SIRT1在乳腺癌组织中和乳腺纤维腺瘤组织中的阳性表达分别为:62.9%(44/70)、35%(7/20),P＜0.05;SIRT1的表达与乳腺癌的病理分期及淋巴结转移有关,P＜0.05;SIRT1表达阳性和阴性患者2年无疾病进展生存率分别为40.6%和73.7%,P＜0.05。结论 乳腺癌组织中SIRT1的阳性表达率较高,并与乳腺癌的发生、发展、侵袭、转移、预后有关。

Objective To investigate the sirtuin (SIRT1) expression in breast cancer and its relationship with clinicopathological parameters in breast tissue. Methods We used immunohistochemical staining (Envision two-step) to detect the expression of 70 cases of breast cancer and 20 cases of breast fibroadenoma of SIRT1. Results The positive expression of sirt1 in breast cancer and breast fibroadenoma tissues were 62.9 % (44/70) and 35 % (7/20),P<0.05. The expression of sirt1 was correlated with the pathological stage and lymph node metastasis of breast cancer, P<0.05. The 2-year survival rates of sirt1 positive and negative patients were 40.6 % and 73.7 %, respectively, P<0.05. Conclusion The positive expression rate of sirt1 in breast cancer is higher, and is related to the occurrence, development, invasion, metastasis and prognosis of breast cancer.

目的 探讨肺淋巴管肌瘤病(PLAM)的临床特征,诊断和治疗,提高对PLAM的认识,合并顽固性低氧血症时需警惕并发肺动脉栓塞,预防漏诊及误诊。方法 回顾分析1例确诊的PLAM合并肺栓塞患者病例资料,并结合相关文献进行复习。结果 PLAM是以弥漫性肺囊性病变为特征的一种罕见的缓慢进展的肿瘤性疾病,好发于育龄期女性,临床表现为呼吸困难、反复自发性气胸和乳糜胸。肺功能多表现为阻塞性或混合性通气功能障碍和弥散功能障碍,动脉血气显示低氧血症。高分辨CT(HRCT)示双肺弥漫性薄壁囊性改变。病理学检查示肺组织淋巴管增生和扩张,管外平滑肌细胞明显增生。诊断主要依靠高分辨CT、肺功能以及病理组织活检。血管内皮生长因子D是无创且可靠的血清学诊断标准。雷帕霉素抑制剂如西罗莫司科可有效治疗本病。结论 育龄期妇女,如反复出现自发性气胸、活动后呼吸困难,胸部HRCT示双肺弥漫分布囊状改变,临床上应想到PLAM可能。随疾病的进展出现顽固性的呼吸困难、低氧血症不能纠正时,需考虑是否合并存在肺动脉栓塞的可能,CT肺动脉造影可确诊。

目的 探讨ERCC1、RRM1、TS蛋白表达对晚期非小细胞肺癌(NSCLC)个体化治疗的指导意义。方法 收集经病理确诊的晚期NSCLC患者87例,其中67例愿意接受药敏免疫组化检测的患者作为研究组,采用SP法检测肿瘤组织ERCC1、RRM1、TS蛋白表达,并根据蛋白表达情况选择化疗方案;另外20例患者不进行药敏免疫组化检测,以常规吉西他滨联合顺铂方案化疗,以此作为对照组。比较两组患者化疗的有效率,疾病控制率(DCR),并以无进展生存期(PFS)为指标比较患者预后。结果 研究组67例患者中,PR 33例(49.25%),SD 13例(19.4%),PD 21例(31.35%);对照组20例患者中,PR 4例(20%),SD 4例(20%),PD 12例(60%),两组疗效之间有差异( χ²=6.437,P=0.04),研究组DCR为68.6%,高于对照组DCR 40%,差异有统计学意义(χ²=5.372,P=0.034)。研究组患者的中位PFS高于对照组,研究组的PFS为5月,对照组为3月,差异有统计学意义(P＜0.05)。结论 对晚期NSCLC患者进行ERCC1、RRM1、TS药敏蛋白免疫组化检测,指导个体化治疗方案,能提高患者化疗的疾病控制率及延长患者的疾病进展时间。

目的 了解异鼠李素对LPS刺激下THP-1细胞炎症因子IL-6、MCP-1、TNF-α释放的影响及其抗炎特征。方法 用PI单染色法检测其细胞存活率;用ELISA法检测THP-1细胞炎症因子IL-6、MCP-1、TNF-α释放。结果 不同浓度的LPS均能使炎症因子IL-6释放增高,且24 h及48 h间水平无差异,但随着LPS刺激浓度的增高,细胞的坏死数量也会随之升高;不同浓度的异鼠李素能在不同时间点不同程度地抑制炎症因子MCP-1、TNF-α、IL-6的释放。结论 异鼠李素能抑制LPS刺激下THP-1细胞炎症因子IL-6、TNF-α、MCP-1的释放,并呈浓度依赖性。

Objectives Our study is aim to investigate the influence of the isorhamnetin on the releasing of inflammatory factor cytokines of the THP-1 cells. Methods PI single staining method was used to detect the cell survival rate. ELISA was used to detecte the concentrations of inflammatory cytokines(MCP-1、IL-6、TNF-α). Results The concentration of IL-6 in THP-1 cells were increased after stimulated with LPS and there no difference between the 24 h group and 48 h group. The survival rate of THP-1 cells decreased as the concentration of LPS increased.The isorhamnetin in different concentration could inhibit the secretion of inflammatory cytokines in different time. Conclusion It is found in our study that isorhamnetin may inhibit the secretion of MCP-1, IL-6, TNF-α in THP-1 cells stimulated by LPS in a concentration dependent way.

目的 探究S100B蛋白、神经元特异性烯醇化酶与新生儿低血糖脑损伤的诊治相关性。方法 收集2014年1月—2016年12月来我院就诊出现低血糖脑损伤的新生儿116例,设为患病组,首先根据临床表现,分为两组,有低血糖症状组(n=54)和无低血糖症状组(n=62)。两组患儿均给予常规药物治疗,有效患者82例,为有效组,无效患者34例,为无效组。同期收集健康足月的新生儿53例,为健康对照组。患儿均于治疗前、后检测血清S100B蛋白、神经元特异性烯醇化酶水平,健康对照组新生儿也于同一时间点进行相同检测。观察各组新生儿血清S100B蛋白、神经元特异性烯醇化酶水平变化,并探究其水平变化与诊断及治疗效果的相关性。结果 低血糖症状组和无低血糖症状组患儿的血糖水平均低于和健康对照组(P<0.05);低血糖症状组患儿的低血糖扶持续时间高于无低血糖症状组(P<0.05)治疗后,各组的NSE和S100B的蛋白水平差异无统计学意义(P>0.05)。患儿血清NSE和S100B与血糖水平呈负相关(r=-0.131、-0.124、P<0.05),与低血糖持续时间呈正相关(r=0.135、0.129,P<0.05)。结论 血清NSE及S100B与患儿血糖水平相关,可作为新生儿低血糖脑损伤的早期诊断指标。血清NSE及S100B水平与治疗效果存在相关性并为负相关,血清水平越低,患者治疗效果越好。

目的 探讨二甲双胍和胰高糖素样多肽-1对2型糖尿病患者并发骨折恢复的影响。方法 选取2016年5月—2017年4月我院骨科收治的2型糖尿病并发骨折患者120例,按随机原则分为5组,每组24例,单药低剂量二甲双胍组(A1)、单药高剂量二甲双胍组(A2)、单药GLP-1组(B)、低剂量二甲双胍联合GLP-1组(C1)和高剂量二甲双胍联合GLP-1组(C2)。二甲双胍低剂量用药量为0.5 g/次,每日2次口服,高剂量用药量为0.5 g/次,每日4次口服。皮下注射利拉鲁肽每日1次,起始量为每日0.6 mg,1周增加为每日1.2 mg,再1周后增加为每日1.8 mg。血糖控制在理想水平后按照标准的手术方法和规程行相应的手术治疗。同时给予饮食控制及其它对症治疗。分别在1、3、6个月时检测其股骨颈骨密度值(BMD)和Harris系统评分。结果 随着治疗时间延长,A1组、C1组、C2组BMD值和Harris系统评分均增高, 在术后3月和6月时,C1组骨密度值和Harris评分高于A1组(P<0.05), C1组骨密度值和Harris评分高于C2组(P<0.05)。结论 胰高糖素样多肽-1可促进2型糖尿病患者骨折愈合、功能恢复,且与低剂量二甲双胍联用促进骨折愈合效果优于与高剂量二甲双胍联用。

Objective To investigate the effects of metformin and glucagon like polypeptide -1 on fracture recovery in patients with type 2 diabetes mellitus(DM). Methods We selected 120 patients with type 2 diabetes mellitus from May 2016 to April 2017 in department of orthopedicsin in our hospital and randomly divided them into 5 groups, 24 cases in each group,includingthe low dose of metformin monotherapy group (A1), the high dose of metformin monotherapy group (A2), single drug GLP-1 group (B), and GLP-1 group low dose of metformin combination (C1) and high dose of metformin combination with GLP-1 group (C2). The low dose of metformin was 0.5 g / time, 2 times a day for oral administration. The high dose was 0.5 g / time, 4 times a day. Subcutaneous injection of liraglutide was once daily, starting at a daily dose of 0.6 mg, 1.2 mg daily after 1 week and 1.8 mg daily after another week. After an ideal level of blood glucose control, corresponding surgical procedures should be performed according to standard surgical methods and procedures. Diet control and other symptomatic treatments were also given. The femoral neck bone mineral density (BMD) and the Harris system score were examined at the first, third, and sixth month respectively. Results With the prolongation of treatment time, the BMD value and Harris system score in the A1 group, C1 group, C2 group were increased. After surgery in March and June, the BMD and Harris score of C1 group were higher than that of A1 group (P<0.05). The bone mineral density and Harris score of C1 group was significantly higher than that of group C2 (P<0.05). Conclusion Glucagon like peptide -1 may promote the fracture recovery and functional recovery in patients with type 2 diabetes mellitus, and with combination of low dose metformin is more effective than that with high dose metformin.

目的 研究孟鲁斯特治疗哮喘的临床疗效及其对患者Th1/Th2细胞免疫平衡和相关炎症因子水平的影响。方法 选取哮喘患者68例随机分为对照组31例及观察组37例,对照组予以常规糖皮质激素吸入治疗,观察组在此基础上加服孟鲁斯特。用药3个月后流式细胞仪检测两组患者外周血Th1、Th2数量变化,ELISA定量外周血IL-4及IFN-γ含量变化并作临床效果评定。结果 两组患者经治疗后,外周血Th1/Th2比例均升高,IL-4/IFN-γ细胞因子水平下降,但观察组较对照组变化更为明显,差异有统计学意义(P＜0.01)。疗效方面,观察组总有效率94.59%,高于对照组的74.19%,差异具有统计学意义(P＜0.01),同时两组均未见明显不良反应。结论 哮喘患者加用孟鲁斯特具有明显免疫调节功能,使Th1/Th2水平趋于平衡,作为哮喘辅助治疗药物疗效确切。

目的 构建吉西他滨耐药乳腺癌细胞4T1耐药株并建立裸鼠乳腺癌肝转移模型。方法 采用低浓度加量持续诱导法,诱导吉西他滨耐药乳腺癌细胞4T1耐药株,命名为4T1/Gem;CCK-8法测定4T1与4T1/Gem细胞的增殖抑制率,计算耐药指数; Western blot法检测细胞P-gp蛋白表达;B超引导下注射4T1/Gem细胞悬液诱导裸鼠肝脏成瘤;HE染色观察肿瘤组织病理情况,免疫组化法检测瘤组织ER、PR、HER2、Ki-67和P-gp蛋白的表达。结果 经过14个月的诱导成功建立4T1/Gem细胞株,可在含40 μg/mL的Gem培养液中稳定生长。4T1/Gem细胞耐药指数为4T1细胞的788.547倍。与亲代相比,4T1/Gem处于G1期和G2期的细胞增加,S期细胞减少;上调P-gp蛋白的表达。4T1/Gem细胞成功建立裸鼠乳腺癌肝转移模型,瘤组织中ER、PR、HER2蛋白阴性表达,Ki-67阳性10％和P-gp蛋白阳性表达。结论 成功构建吉西他滨耐药乳腺癌细胞4T1耐药株并建立裸鼠乳腺癌肝转移模型,为开发治疗乳腺癌肝转移化疗耐药的药物提供实验基础。

Objective To construct a gemcitabine-resistant variant of the breast cancer cell line (4T1/Gem) and establish a nude mouse model of breast cancer with hepatic metastatic. Methods A gemcitabine-resistant variant of the breast cancer 4T1 cell line was induced by gradually increasing the concentration of gemcitabine; this variant is referred to in this study as 4T1/Gem. The proliferation suppression rates of 4T1 and 4T1/Gem cells were determined by using the CCK-8 essay to evaluate the drug resistance indices of the cell lines. Western blot analysis was used to detect P-gp protein expression. Under ultrasonography, a 4T1/Gem cell suspension was injected into nude mice to induce liver tumors. H&E staining was used to observe tumor pathology, and immunohistochemistry was used to detect the expression of ER, PR, HER-2, Ki-67, and P-gp. Results After 14 months of induction, a 4T1/Gem cell line is established successfully. The cell line can grow stably in culture liquid containing 40 μg/ml gemcitabine. The drug resistance index of 4T1/Gem is 788.547. Compared with the 4T1 cell line, the 4T1/Gem cell line can upregulate P-gp protein expression and successfully establish a nude mouse model of breast cancer with hepatic metastatic. ER, PR, and HER-2 proteins exhibit negative expression in the tumor tissue. The positive expression of P-gp and 10% of Ki-67 proteins is also observed. Conclusion This study successfully constructs a gemcitabine-resistant variant of the breast cancer cell line (4T1/Gem)and establishes a nude mouse model of breast cancer with hepatic metastatic, thereby providing an experimental basis for developing and treating a drug-resistant variant of breast cancer.