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 探讨黄连素联合左氧氟沙星对肺炎克雷伯菌(KPn)抑菌作用。方法 KPn分为敏感株组和耐药株组,采用琼脂二倍稀释法测定黄连素联合左氧氟沙星对KPn的最低抑菌浓度(MIC)、抑制99％接种细菌生长的最低抑菌浓度(MIC99)、防突变浓度(MPC);比浊法测定黄连素联合左氧氟沙星对KPn生长曲线的影响。结果 与单用左氧氟沙星相比,联合黄连素后,敏感株组和耐药株组对左氧氟沙星MIC的下降率分别为33.3%和20%,2组之间无统计学差异(P>0.05)。左氧氟沙星与50 μg/mL黄连素或500 μg/mL黄连素联用后的抗菌能力均较单用左氧氟沙星好,且高浓度黄连素的联合抑菌效果较低浓度更加明显,差异有统计学意义(P<0.05)。与50 μg/mL黄连素联用后,左氧氟沙星SI下降了1/5 ;而与500 μg/mL黄连素联用则下降3/5 。结论 本实验证明了黄连素与左氧氟沙星联用能增加左氧氟沙星对KPn的抗菌作用,可以明显缩小耐药突变选择窗(MSW),且高浓度黄连素联合抗菌作用较低浓度好。

Objective To explore antibacterial effect of berberine(Ber) on K. Pneunmoniae(KPn) combing with levofloxacin(LVX). Methods KPn was divided into sensitive and resistant strains groups.The MIC, MIC99 and MPC of Ber combing with LVX on KPn was determined by the agar dilution method.The growth curve of Ber combing with LVX on KPn was measured by turbidimetry. Results Ber combined with LVX compare with LVX alone, MIC descent rate of sensitive strains group was 33.3%, resistant strains group was 20%, and there were no statistical differences along two groups(P>0.05). Ber combined with LVX could increase antibacterial effect and high concentration was more obvious than the low one, there were statistical differences(P<0.05). Compared with LVX alone,the SI value of 50 μg/mL Ber combined with LVX was decreased 1/5, and the SI value of 500 μg/mL Ber combined with LVX was decreased 3/5. Conclusion Ber combing with LVX could increase bacteriostatic effect on KPn,and reduce MSW significantly; high concentration of berberine was better than low one.

目的 探讨原发性肉碱缺乏症的诊断与治疗方案,对2例原发性肉碱缺乏症患儿及其家系行SLC22A5基因检测,确定基因突变位点,为家系提供遗传疾病的咨询。方法 用串联质谱技术对1例疑似患儿进行游离肉碱及多种酰基肉碱检测,对游离肉碱降低的患儿行SLC22A5基因突变检测,确诊PCD,对其姐姐行上述检查。对2例确诊PCD患儿补充左旋肉碱治疗,随访11个月。并对其家系行SLC22A5基因检测。结果 2例确诊PCD患儿,1例为临床患儿,另1例为其姐姐,无明显临床表现。2例患儿均检测到基因突变。2例患儿血游离肉碱水平低于参考值,伴多种酰基肉碱显著降低,均给予补充左旋肉碱治疗,1例治疗2月后症状改善,另1例未曾未发病,血游离肉碱及其他酰基肉碱水平上升至正常。2例患儿SLC22A5 c.760C>T,(p.Arg254X)纯合,致病突变;患儿父母亲SLC22A5基因的c.760C位点检测,发现:均携带c.760C>T,(p.Arg254X)杂合突变。结论 应用串联质谱技术检测血游离肉碱、多种酰基肉碱水平及SLA22A5基因突变检测诊断了2例PCD,均补充左旋肉碱取得较好疗效。SLC22A5基因c.760C>T,(p.Arg254X)突变是本家系中患有PCD的致病突变,用错义突变和剪切改变的分析手段对SLC22A5基因的外显子编码区进行直接测序可为PCD家系提供遗传咨询。

Objective To explore the diagnosis and treatment of primary carnitine deficiency. To identify potential mutation of SLC22A5 gene in two children affected with primary carnitine deficiency and provide genetic counseling. Methods We measured the free camitine(Co)and acylcamitine levels in a suspected clinical inherited metabolic diseases by tandem mass spectrometry. The SLC22A5 gene mutations were tested to the children with low Co level and the diagnosis was made. Then, We measured the free camitine(Co)and acylcamitine levels and SLC22A5 gene mutations in her sister. The children with PCD were treated with carnitine and followed up for 11 months. The SLC22A5 gene was detected in their family. Results In two children affected with PCD, 1 case was clinical children, another case of their sister was no obvious clinical manifestations. Mutations were found in all of them.The average C0 level in patients was lower than the reference value,along with decreased level of different acylcamitines. Two cases were treated with earnitine. Their clinical symptoms reduced 2 months later. Another case had not been sick. The CO level and different acylcamitines level in the blood rose to normal. A homozygous mutation C. 760C>T (P. Arg254X)of the SLC22A5 gene was detected in the two cases.Heterozygous mutation C. 760C>T (P.Arg254X) was also found in other family members. Conclusion Two patients were diagnosed with PCD by the test levels of free carnitine and acylcarnitines in blood with tandem mass spectrometry,and gene mutation test. L-carnitine supplement had a good effect in treatment of the PCD patients.C.760C> T (P.Arg254X) mutations of the SLC22A5 gene is the deleterious mutations for PCD families, The analysis method of the wrong mutagenesis and shear changes which is used to directly sequence the exons codes of the SLC22A5 gene can provide genetic counseling for PCD families.

目的 观察紫河车提取物联合顺铂对人脑胶质瘤细胞增殖与凋亡的影响。方法 把正常培养传代后的U251胶质瘤细胞按随机分配的方法分为四组,A组仅加普通培养液,B、C、D组各加紫河车提取物(400 mg/mL)2 mL、顺铂(1 mg/mL)0.01 mL、紫河车提取物(400 mg/mL)2 mL＋顺铂(1 mg/mL)0.01 mL;MTT法观察U251细胞增殖情况,流式细胞仪检测U251细胞凋亡率。结果 培养12、24、36、48、60 h,B、C、D组细胞增殖指数逐渐下降,与A组进行比较,各组P值均小于0.05;其中,将D组与B、C组进行比较,P值小于0.05。将各组培养24 h后上机,测得A、B、C、D各组细胞的凋亡率分别为(0.3±0.2)%,(10.6±1.5)%,(35.9±2.8)%,(52.1±4.1)%。其中,B、C、D各组和A组进行比较,P值均小于0.05;将D组与B、C组两组进行比较,P值也均小于0.05。结论 紫河车提取物联合顺铂可抑制人脑胶质瘤U251细胞增殖,并诱导其凋亡。

Objective To observe the effect of cisplatin combinated with the placental immunoregulating polypeptide (PIP) on proliferation and apoptosis of glioma cells. Methods Randomly we divide the normal handed U251 glioma cells into four groups. We added ordinary nutrient solution to group A, while added activated PIP(400 mg/mL)2 mL to group B, cisplatin (1 mg/ml) 0.01 ml to group C, PIP 400 mg/mL)2 mL and cisplatin (1 mg/mL) 0.01 mL to group D. We surveyed the proliferation rate of gliobma cells by MTT experimental method and analyzed the apoptosis of U251 glioma cells by flow cytometry. Results The index of cell proliferation of group B,C,D declined gradually with the training of 12 h,24 h,36 h,48 h,60 h. Compared B, C,D group with A group, P<0.05,and compared group D with group B and group C, P< 0.05. Put groups culturing of 24 hour on flow cytometer, the glioma cells apoptosis rate of each group was 0.3%±0.2%、10.6%±1.5%、35.9%±2.8%、52.1%±4.1% respectively. Compared group B,C,D with group A, P<0.05,and compared group D with group B and group C, P<0.05. Conclusion Placental immunoregulatingpPolypeptide combined with cisplatin may restrain the proliferation of human glioma cells, meanwhile increase the apoptosis of glioma cells.

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 探讨川芎嗪对链脲佐菌素(STZ)诱导2型糖尿病大鼠肾病的治疗作用及机制。方法 SD大鼠50只,随机分为正常组和模型组。除正常组外,其余大鼠均给予高脂-高糖饲料喂养4周,再给予链脲佐菌素(40 mg/kg,ip),72 h后测定空腹血糖,将血糖值高于16.67 mmol/L的大鼠随机分成4个组即模型组,二甲双胍阳性组(250 mg/kg),川芎嗪低、高剂量组(80、160 mg/kg),连续给予相应试药8周。其中正常组和模型组的大鼠均给予同等量蒸馏水灌胃。实验结束时,测定大鼠血糖、尿蛋白、血尿素氮和血肌酐含量;免疫组化法测定大鼠肾组织TLR4和caspase3蛋白表达。光镜下观察肾脏病理学变化。结果 与模型组比较,二甲双胍组和川芎嗪高剂量组给药8周后,大鼠动态空腹血糖均能明显降低(P<0.05),大鼠动态尿蛋白显著性降低(P<0.01,P<0.05); 二甲双胍和高剂量组TLR4和caspase3蛋白表达明显低于模型组(P<0.05);肾脏组织病理性损伤明显减轻。结论 川芎嗪对STZ诱导2型糖尿病大鼠肾病具有保护作用,其机制可能与下调TLR4表达作用有关。

Objective To investigate the therapeutic effects and mechanisms of tetramethylpyrazine (TMP) on streptozocin(STZ)-induced-nephropathy in type 2 diabetic rats. Methods 50 SD rats were randomly divided into normal group(n=10) and model group(n=40). The model rats were fed on high fat and sugar diets for 4 weeks, then given STZ(40 mg/kg,ip). After 72 hours, the fasting blood glucose (FBG) was measured. Rats with high FBG above 16.67mmol/L were randomly divided into four groups: model, metformin(Met, 250 mg/kg)and TMP (80 mg/kg, 160 mg/kg) groups for treating 8 weeks, and both the control and model groups were given equals distilled water by intragastric administration. At the end of the experiment, blood glucose, urine protein, blood urea nitrogen and creatinine were measured. The expression of TLR4 and caspase3 protein in kidney tissue of rats was determined by immunohistochemistry. Pathological changes of kidney were observed under light microscope. Results Compared with the model group, metformin and high dose of TMP administered after 8 weeks, rats can significantly reduce the dynamic fasting blood glucose(P<0.05). Urinary protein excretion of total dynamic decreased significantly (P<0.01, P<0.05); the protein expression of TLR4 and caspase3 in the metformin group and high dose group was significantly lower than that in the model group (P<0.05); kidney tissue pathological damage was significantly reduced. Conclusion TMP has a protective effect on STZ induced nephropathy in type 2 diabetic rats, and its mechanism may be related to the down-regulation of TLR4 expression.

目的 探索长链非编码RNA LINC00672在肺癌组织中的表达及其与患者预后的关系。方法 采用实时荧光定量PCR技术检测LINC00672在75对肺癌组织和癌旁正常组织中的表达,分析其在癌组织中的表达水平与肺癌患者临床分期和预后的关联。结果 LINC00672在肺癌组织中的表达显著低于癌旁正常组织(P=0.026),LINC00672高表达与低表达相比能显著降低肺癌患者的死亡率(死亡风险比=0.46;95%置信区间=0.23~0.95;P=0.036),延长患者中位生存期(34个月 vs 18个月,P=0.027)。并且,LINC00672与肺癌预后的关联在低年龄组(<60 a)、吸烟者和非饮酒者中更为显著。进一步相乘交互作用分析显示LINCOO672与饮酒在肺癌死亡风险上具有显著的交互效应(P=0.049)。然而,LINC00672的表达水平在不同分期、T、N、M患者来源的肺癌组织中的表达无显著性差异。结论 LINC00672与肺癌发生发展存在关联,可用于预测肺癌患者的预后。

Objective To explore the expression status of long non-coding RNA LINC00672 in lung cancer tissues and its correlation with survival of lung cancer. Methods We applied the real-time PCR method to measure the expression level of LINC00672 in 53 pairs of lung cancer tissues and adjacent lung normal tissues, and analyzed the correlation between its expression and survival of lung cancer. Results LINC00672 was significantly down-regulated in lung cancer tissues than their adjacent lung normal tissues (P=0.026). Compared to those with low expression level of LINC00672, patients with high expression level of LINC00672 exerted a significant long median survival time than those with low expression level (34 vs 18 months, P=0.027). High LINC00672 expression also contributed to low mortality rate than low LINC00672 expression (hazard ratio=0.46, 95% confidence interval =0.23-0.95, P=0.036). Meanwhile, the correlation was more evident in those low age groups (< 60 years), smokers and non-drinkers. There was also a significant interaction between LINC00672 and drinking on affecting death risk of lung cancer. However, no significant association was observed between LINC00672 expression and clinical stages as well as T, N, M status. Conclusion LINC00672 is correlated with development of lung cancer, which may be a valuable biomarker to predict lung cancer prognosis.

目的 分析PBK在前列腺癌中的表达及临床意义。方法 利用前列腺癌的组织芯片,包含98例前列腺癌及81例对照癌旁组织作为研究对象,免疫组化方法检测PBK的表达情况,并运用统计学方法分析免疫组化芯片及Taylor数据库中PBK表达与前列腺癌临床病理特征之间的关系。结果 PBK在前列腺癌中表达明显升高(P=0.001);且在Gleason高评分组的表达比低评分组表达升高(P=0.001)。Taylor数据库得到相似结果,且运用Kaplan-Meier分析发现PBK与无生化复发生存率显著相关(P=0.007),最后采用Cox回归模型进行多因素综合分析发现在影响前列腺癌预后的队列中,PBK高表达(P=0.041)与Gleason评分、病理分期都是前列腺癌生化复发的独立预测指标。结论 PBK的表达与前列腺癌密切相关,可作为临床诊断及治疗的分子标志物。

Objective To investigate the expression and clinical significance of PBK in prostate cancer. Methods Using tissue microarrays of prostate cancer, which including 98 cases of prostate cancer and 81 cases of normal tissue adjacent to cancer as the research object, the expression of PBK was detected by immunohistochemistry, and statistical analysis was used to analyze the relationship between the expression of PBK and the clinicopathological features of prostate cancer in the microarray and Taylor database. Results The expression of PBK in prostate cancer was significantly higher (P=0.001), and the expression increased in the group of high Gleason score (P=0.001). The Taylor database obtained similar results, and Kaplan-Meier analysis showed that PBK was significantly correlated with the biochemical recurrence free survival (P=0.007). Finally, Cox regression model was used to analyze the prognostic factors of prostate cancer. Result shows that, the high expression of PBK (P=0.041), Gleason score and pathological stage were independent predictors of biochemical recurrence of prostate cancer. Conclusion The expression of PBK is closely related to prostate cancer, and can be used as a molecular marker for clinical diagnosis and treatment.

成人急性呼吸窘迫综合征死亡率居高不下,而当前治疗手段大多以对症支持为主且效果欠佳。通过早期识别ARDS高危患者,及早施加干预措施阻断疾病进展是目前研究的新方向。较之传统机械通气模式,气道压力释放通气因其特有的肺保护作用备受国内外学者关注,APRV下肺泡持续开放并保持稳定可减少肺损伤发生,将APRV应用于ARDS预防理论上前景可观,本文将围绕早期实施APRV对预防ARDS应用价值展开论述。

The mortality of adult acute respiratory distress syndrome(ARDS) remains high. Supportive care measures are main treatments to ARDS currently while the effect is poor. Early application of airway pressure release ventilation in patients who are at high risk for ARDS is a new direction of research. Compared with traditional mechanical ventilation, more and more scholars both at home and abroad pay attention to the airway pressure release ventilation APRV because of its unique lung protection. Under APRV the alveoli maintain stable continuously which may reduce the incidence of lung injury. Applying APRV to ARDS prevention is promising theoretically. This paper will focus on the value of preemptive airway pressure ventilation for high-risk ARDS patients.