rhCG融合蛋白在CHO细胞中的表达、纯化与鉴定

为实现rhCG融合蛋白在CHO细胞中的高效表达,构建两个不同的 His－tag rhCG 融合蛋白真核表达载体,通过瞬时转染比较两个载体在CHO－dhfr－细胞中的表达量,选择表达量较高的载体稳定转染CHO－dhfr －,并采用氨甲喋呤（ MTX）进行加压筛选高表达单克隆细胞株。加压后,表达量由1日1300mIU? mL －1提高至18000mIU? mL －1（以106个细胞计）。该克隆株经传代50代以后,rhCG表达量未发生明显变化,表明该克隆为稳定克隆株。用Ni＋螯合亲和层析及凝胶过滤层析分离纯化表达产物,获得纯度大于99％的rhCG融合蛋白,SDS－PAGE电泳及 Western Blot结果表明rhCG得到正确及完整的表达,生物学活性测定结果表明样品比活为11000IU? mg －1。     

Microvascular heart involvement in systemic autoimmune diseases: The purinergic pathway and therapeutic insights from the biology of the diseases

Heart involvement – often asymptomatic – is largely underestimated in patients with systemic autoimmune diseases (SADs). Cardiovascular events are more frequent in patients with SADs compared to the general population, owing to the consequences of inflammation and autoimmunity and to the high prevalence of traditional risk factors. Coronary microvascular disease (CMD) is a form of cardiac involvement that is increasingly recognised yet still largely neglected. CMD, the incapacity of the coronary microvascular tree to dilate when myocardial oxygen demand increases or when there is a microvascular spasm (or subclinical myocarditis), is increasingly reported because of the widespread use of new cardiac imaging tools, even in a subclinical phase. The assessment of myocardial coronary flow reserve (CFR) emerged as the most effective clinical tool to detect microvascular damage. The potential causes of microvascular damage, molecular and cellular inflammation along with a pathological CD39-CD73 axis, need always to be considered because data show that they play a role in the occurrence of acute coronary syndromes, heart failure and arrhythmias, even in the early asymptomatic stage. Data suggest that controlling disease activity by means of methotrexate, biologic drugs, antimalarial medications, statins and aspirin, according to indication, might reduce the cardiovascular risk related to macrovascular and microvascular damage in most patients with SADs, provided that they are used early and timely to control diseases. The need of new biomarkers and a careful assessment of myocardial CFR emerged as the most effective clinical tool to detect microvascular damage.     

Inhibiting homologous recombination decreases extrachromosomal amplification but has no effect on intrachromosomal amplification in methotrexate-resistant colon cancer cells

Gene amplification, which involves the two major topographical structures double minutes (DMs) and homegeneously stained region (HSR), is a common mechanism of treatment resistance in cancer and is initiated by DNA double-strand breaks. NHEJ, one of DSB repair pathways, is involved in gene amplification as we demonstrated previously. However, the involvement of homologous recombination, another DSB repair pathway, in gene amplification remains to be explored. To better understand the association between HR and gene amplification, we detected HR activity in DM- and HSR-containing MTX-resistant HT-29 colon cancer cells. In DM-containing MTX-resistant cells, we found increased homologous recombination activity compared with that in MTX-sensitive cells. Therefore, we suppressed HR activity by silencing BRCA1, the key player in the HR pathway. The attenuation of HR activity decreased the numbers of DMs and DM-form amplified gene copies and increased the exclusion of micronuclei and nuclear buds that contained DM-form amplification; these changes were accompanied by cell cycle acceleration and increased MTX sensitivity. In contrast, BRCA1 silencing did not influence the number of amplified genes and MTX sensitivity in HSR-containing MTX-resistant cells. In conclusion, our results suggest that the HR pathway plays different roles in extrachromosomal and intrachromosomal gene amplification and may be a new target to improve chemotherapeutic outcome by decreasing extrachromosomal amplification in cancer.     

How much progress has there been in the second-line treatment of multiple sclerosis: A 2017 update

In 1993, the US Food and Drug Administration (FDA) approved the first drug specifically for treating multiple sclerosis (MS). More than two decades later, a dozen such treatments are now available. Of these, four are considered second-line treatments for use in escalation strategies and two new drugs are currently undergoing accreditation procedures. Soon, they will provide clinicians with a range of six effective disease-modifying treatments (DMTs) to thwart the inflammatory processes in MS patients with active disease. However, while such a large number of DMTs for MS can help to control early inflammation, any decisions to be made by clinicians have also been made substantially more complex. This complexity is increased by the lack of head-to-head studies comparing these second-line therapies and the benefit–risk profiles for each of these drugs, which are likely to vary among patients. Ultimately, good awareness of the benefits and, more important, the risks of each MS DMT is crucial for the effective management of inflammation in MS.     

联合用药对大剂量甲氨蝶呤化疗治疗儿童急性淋巴细胞白血病肾毒性及血药浓度的影响

目的 探讨联合用药对大剂量甲氨蝶呤（MTX）化疗肾毒性及MTX血药浓度的影响。方法 采用回顾性分析方法,收集2012年9月至2014年9月广西医科大学第一附属医院178例儿童急性淋巴细胞白血病（ALL）患者共633例次大剂量MTX化疗的临床资料,根据化疗时用药情况分为两组,对照组为常规大剂量MTX化疗,联合用药组在此基础上联用了青霉素类、非甾体类抗炎药及质子泵抑制剂等,比较两组的急性肾损伤（AKI）发生率和MTX血药浓度。结果 有84例次大剂量MTX化疗时联合应用了哌拉西林、布洛芬和奥美拉唑等药物,联合用药组AKI总体发生率明显高于对照组（P<0.05）,表现在中度和重度AKI发生率明显增加（P<0.05）。联合用药组各时间点MTX血药浓度明显高于对照组（P<0.05）,MTX血药浓度降至安全范围所需天数明显长于对照组（P＜0.05）。结论 大剂量甲氨蝶呤化疗时联用哌拉西林、布洛芬和奥美拉唑等药物会使MTX排泄延缓,肾毒性发生风险增加,应避免联用。     

腹腔镜手术联合甲氨喋呤治疗异位妊娠的疗效观察

目的 探讨腹腔镜手术联合甲氨喋呤治疗异位妊娠的疗效.方法 将2010年5月至加10年10月在广州市妇婴医院确诊为异位妊娠的56例患者随机分成两组.观察组:45例,采用腹腔镜手术联合甲氨喋呤(MTX),对照组:11例,单采用腹腔镜手术.比较两组患者治愈成功率、治疗前、后血β-HCG下降值及两种手术的住院时间.结果 两组患者治愈成功率比较差异无统计学意义(P>0.05),观察组血β-HCG降至正常的时间明显快于对照组(P<0.05),两组术后的输卵管通水情况及两组住院天数比较差异无统计学意义(P>0.05).结论 采用腹腔镜手术联合甲氨喋呤治疗异位妊娠能更快、更有效地抑制滋养细胞增长,提高疗效,缩短治疗时间,减少治疗费用.     

异位妊娠保守治疗不同方案疗效分析

目的：探讨异位妊娠保守治疗的不同治疗方案及其疗效的比较。方法：对120例未破裂型异位妊娠病例进行治疗观察,随机分成3组：A组采用MTX50mg／m^2;B组采用MTX50mg／m^2;C组为B组方案的同时加用米非司酮25mg。结果：A组成功率为72．50％（29／40）;B组成功率82．50％（33／40）;C组成功率为80．00％（32／40）;A组与B、C组间均有显著性差异（P〈0．05）;B、C组间无显著性差异（P〉0．05）。结论：使用MTX50mg／m^2单次im,较分次肌内注射加（四氢叶酸）CF解救方案疗效好,而加用米非司酮方案并未增加MTX治疗的成功率,反而相应地增加了药物的不良反应。     

氨甲喋呤联合妇科千金胶囊治疗异位妊娠80例临床观察

[目的]评价氨甲喋呤联合妇科千金胶囊治疗异位妊娠的临床疗效。[方法]80例临床诊断为异位妊娠可以保守治疗的患者,随机分为两组,治疗组(A组)50例,氨甲喋呤联合妇科千金胶囊治疗;对照组(B组)30例,单纯氨甲喋呤治疗。比较各组治愈率及输卵管通畅情况。[结果]A组成功率为93.29%,B组为89.79%,两组成功率差异无统计学意义,但治疗后包块消失时间差异有统计学意义(P<0.05),随访输卵管通畅情况两组差异有统计学意义(P<0.05)。[结论]氨甲喋呤联合妇科千金胶囊治疗异位妊娠可以缩短包块消失时间,提高输卵管的通畅率。