人诱导多能干细胞向血管内皮细胞分化方法的建立

目的:建立一种新的促进人诱导多能干细胞(iPSc)向血管内皮细胞(VEC)分化的方法.方法:将未分化的人iPSc制备成拟胚体(EBs),分为常氧组(A组)、常氧加血管内皮生长因子(VEGF)组(B组)、常氧加甲基乙二酰氨基乙酸(DMOG)组(C组)、缺氧组(D组)、缺氧加VEGF组(E组)及缺氧加DMOG组(F组),分别给予相应处理;观察各组iPS-EBs分化的VEC形态结构,应用逆转录PCR(RT-PCR)法检测VEC特异基因CD31、CD34、VE-cad、KDR、vWF的表达,免疫荧光细胞法检测以mRNA表达最强组的VEC特异基因在细胞定位情况判断VEC分化情况.结果:RT-PCR结果显示未经诱导iPSc不表达任何VEC相关基因,A组EBs细胞仅表达微弱的VE-cad和KDR;经条件诱导后iPS-EBs细胞表达CD31、CD34、VE-cad、KDR、vWF,C组表达最强,与其他各组比较差异有统计学意义(P＜0.05);免疫荧光结果显示C组中CD31、CD34为膜表达,KDR、vWF为胞浆表达.结论:常氧状态下加入DMOG培养能成功促进人iPSc向VEC分化.     

桔梗皂苷D对MG-63细胞的凋亡作用

目的 研究桔梗皂苷D对MG-63细胞的促凋亡作用.方法 MTT法测定桔梗皂苷D对MG-63细胞增殖抑制情况及其IC50值,利用流式细胞仪检测加入桔梗皂苷D作用24 h后的MG-63细胞的凋亡情况、凋亡类型.使用二氯荧光黄二乙酸酯(DCFH-DA)检测加入桔梗皂苷D后肿瘤细胞内活性氧水平,加入JC-1线粒体染料试剂盒检测被桔梗皂苷D孵育24 h后肿瘤细胞线粒体膜电位的变化.结果 MTT数据表明桔梗皂苷D对细胞的生长半数抑制浓度IC50为11.80μmol/L.MG-63细胞被桔梗皂苷D作用24 h后,通过JC-1和DCFH-DA染色的实验结果表明桔梗皂苷D作用后的MG-63细胞内的活性氧水平上升,细胞线粒体膜电位下降.结论 桔梗皂苷D可能通过增加细胞内活性氧水平破坏线粒体从而导致细胞发生凋亡.     

胃癌本虚标实辨证的病理生理学基础探讨

通过对胃癌患者临床表现的观察,提出胃癌本虚标实辨证分型。并探讨其病理生理学基础。结果表明:细胞免疫功能下降,主要是 LCT、CD_3、CD_4下降,CD_8升高,CD_4/CD_8比值降低;体液免疫升高趋势,主要是 IgA、补体 C_3、C_4和 B 因子,而 IgM下降。内分泌激素皮质醇升高为主,T_3都下降,脾肾阳虚同时伴 T_4下降。胃肠吸收功能都下降。癌标记物都升高。在此结果的基础上,提出胃癌中医健脾益气,补肾温阳,理气活血,祛痰清热的治法。     

疼痛敏感性与腹腔镜胃肠肿瘤手术患者急性术后疼痛相关性的研究

目的 探讨疼痛敏感性与腹腔镜胃肠肿瘤患者急性术后疼痛的相关性。方法 择期全身麻醉下行腹腔镜胃肠肿瘤手术患者50例,性别不限,年龄≥18岁,ASA分级Ⅰ～Ⅱ级,根据术前疼痛敏感性调查问卷（PSQ）评分结果分为2组：高敏感性组（PSQ≥5.0分,S组）和低敏感性组（PSQ＜5.0分,C组）。采用视觉模拟评分（VAS）评估患者术后24h内急性疼痛的发生情况,记录术前PSQ评分、外周静脉置管疼痛评分（VCP)、年龄、性别、ASA分级、体重指数（BMI）、手术时长、全麻苏醒时间、拔管时间、术后清醒即刻（T0)、1h(T1)、2h(T2)、3h(T3)、4h(T4)、5h(T5)、6h(T6)、8h(T7)、10h(T8)、12h(T9)、24h(T10)的VAS评分、术后24h内镇痛泵按压次数和补救镇痛次数及恶心、呕吐、嗜睡、腹胀等术后并发症的发生情况。结果 两组患者的年龄、性别、ASA分级、BMI、手术时长、苏醒时间、拔管时间、术后24h镇痛泵按压次数、术后并发症比较无统计学差异（P＞0.05）。与C组比较,S组T0、T1、T2时间点的VAS评分较高,有统计学差异（P＜0.05）。术前PSQ评分与VCP呈正相关（r=0.693,P＜0.05）,结果有统计学意义。术前PSQ评分与T0、T1、T2时间点的VAS评分呈正相关（P＜0.05）。S组术后2h补救镇痛次数较C组增多,有统计学差异（P＜0.05）。以术前PSQ评分为检验变量,急性术后疼痛的发生为状态变量绘制ROC曲线。测得曲线下面积为0.909,提示采用PSQ问卷可显著预测腹腔镜胃肠肿瘤手术患者急性术后疼痛的发生。通过约登指数计算出腹腔镜胃肠肿瘤手术患者中PSQ≥4.85分者疼痛敏感性更高,其VCP≥2分的发生率为66.7%,而PSQ＜4.85分的患者中VCP≥2分的发生率仅为24.1%,比较有统计学差异（P＜0.05）。结论 疼痛敏感性与腹腔镜胃肠肿瘤手术患者急性术后疼痛的发生有相关性,可能成为急性术后疼痛发生的预测指标。腹腔镜胃肠肿瘤手术患者中,PSQ≥4.85分者术后发生疼痛的可能性更高。     

基于复杂系统中因果关系的单细胞病理学

肿瘤是由肿瘤细胞和周围的微环境组成的复杂生态系统。在病理学上,肿瘤中细胞各异、分布和生理功能千差万别,并且受自身、周围环境和治疗的调节,自成复杂系统。单细胞分析技术为病理学复杂系统的研究提供了更多的信息。利用复杂系统中的因果判定方法,可以揭示单细胞的复杂的调节作用,以及不同类型的肿瘤细胞与患者治疗方案、治疗效果、疾病复发和生存时间的因果关系。     

Transplanted human cones incorporate into the retina and function in a murine cone degeneration model

Once human photoreceptors die, they do not regenerate, thus, photoreceptor transplantation has emerged as a potential treatment approach for blinding diseases. Improvements in transplant organization, donor cell maturation, and synaptic connectivity to the host will be critical in advancing this technology for use in clinical practice. Unlike the unstructured grafts of prior cell-suspension transplantations into end-stage degeneration models, we describe the extensive incorporation of induced pluripotent stem cell (iPSC) retinal organoid–derived human photoreceptors into mice with cone dysfunction. This incorporative phenotype was validated in both cone-only as well as pan-photoreceptor transplantations. Rather than forming a glial barrier, Müller cells extended throughout the graft, even forming a series of adherens junctions between mouse and human cells, reminiscent of an outer limiting membrane. Donor-host interaction appeared to promote polarization as well as the development of morphological features critical for light detection, namely the formation of inner and well-stacked outer segments oriented toward the retinal pigment epithelium. Putative synapse formation and graft function were evident at both structural and electrophysiological levels. Overall, these results show that human photoreceptors interacted readily with a partially degenerated retina. Moreover, incorporation into the host retina appeared to be beneficial to graft maturation, polarization, and function.     

Rhodiola rosea polysaccharides promote the proliferation of bone marrow haematopoietic progenitor cells and stromal cells in mice with aplastic anaemia

ContextThe effects of Rhodiola rosea L. (Crassulaceae) polysaccharides (RRPs) on haematopoiesis are poorly understood.ObjectiveTo determine the effects of RRPs on haematopoiesis in mice with aplastic anaemia.Materials and methodsAplastic anaemia was induced in Kunming mice by ⁶⁰Coγ (2.0 Gy) irradiation and cyclophosphamide administration (50 mg/kg/day for 3 consecutive days; intraperitoneal injection). The in vivo effects of RRPs (10, 20, and 40 mg/kg; intraperitoneal injection) on haematopoiesis were analyzed using peripheral blood tests, histopathological examination of haematopoietic tissues, culture of haematopoietic progenitors and bone marrow stromal cells (BMSCs), and Western blotting of Fas and Fas ligand (FasL). The in vitro effects of RRPs on bone-marrow haematopoietic progenitors and BMSCs were also evaluated.ResultsCompared to anaemic controls, high-dose RRPs (40 mg/kg) significantly increased red blood cells (8.21 ± 0.57835 versus 6.13 ± 1.34623 × 10¹²/L), white blood cells (5.11 ± 1.6141 versus l.54 ± 1.1539 × 10⁹/L), and BMSCs (10.33 ± 1.5542 versus 5.87 ± 3.1567 × 10¹²/L) in mice with aplastic anaemia (all p < 0.01). High-dose RRPs significantly increased the formation of colony-forming unit-granulocyte macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and colony-forming unit-erythroid (CFU-E; p < 0.01). Fas and FasL protein expression in BMSCs decreased after RRPs administration. Especially at the high dose, RRPs (150 μg/mL) significantly promoted in vitro CFUs-E, BFUs-E, and CFUs-GM formation. RRPs (150–300 μg/mL) also promoted BMSC proliferation.Discussion and conclusionsRRPs helped to promote haematopoietic recovery in mice with aplastic anaemia, facilitating haematopoietic tissue recovery. This study indicated some mechanisms of the haematopoietic regulatory effects of RRPs. Our findings provide a laboratory basis for clinical research on RRPs.     

髓系抑制细胞、Th17细胞在哮喘患儿外周血中的表达

目的 分析髓系抑制细胞(MDSCs)及Th17细胞在哮喘患儿外周血中的表达情况和临床意义.方法 选取自贡市第一人民医院儿科2013年6月至2015年8月收治的120例哮喘患儿作为观察组,另外选择87例健康儿童作为对照组.应用流式细胞术检测MDSCs、Th17细胞在哮喘患儿外周血中的表达情况.结果 和对照组相比,观察组MDSCs占有核细胞的百分比和Th17细胞占单个核细胞的百分比都显著升高(Hc值分别为56.14、45.98,均P<0.05),差异具有统计学意义.结论 从MDSCs、Th17细胞在哮喘患儿外周血中的表达情况来看,它们可能参与了婴幼儿哮喘的发生与发展过程.     

Differential Effects of Dietary versus Exercise Intervention on Intrahepatic MAIT Cells and Histological Features of NAFLD

Background: Mucosal-associated invariant T (MAIT) cells promote inflammation in obesity and are implicated in the progression of non-alcoholic fatty liver disease (NAFLD). However, as the intrahepatic MAIT cell response to lifestyle intervention in NAFLD has not been investigated, this work aimed to examine circulating and intrahepatic MAIT cell populations in patients with NAFLD, after either 12 weeks of dietary intervention (DI) or aerobic exercise intervention (EI). Methods: Multicolour flow cytometry was used to immunophenotype circulating and intrahepatic MAIT cells and measure MAIT cell expression (median fluorescence intensity, MFI) of the activation marker CD69 and apoptotic marker CD95. Liver histology, clinical parameters, and MAIT cell populations were assessed at baseline (T0) and following completion (T1) of DI or EI. Results: Forty-five patients completed the study. DI participants showed decreased median (interquartile range) expression of the activation marker CD69 on circulating MAIT cells (T0: 104 (134) versus T1 27 (114) MFI; p = 0.0353) and improvements in histological steatosis grade post-intervention. EI participants showed increased expression of the apoptotic marker CD95, both in circulating (T0: 1549 (888) versus T1: 2563 (1371) MFI; p = 0.0043) and intrahepatic MAIT cells (T0: 2724 (862) versus T1: 3117 (1622) MFI; p = 0.0269). Moreover, the percentage of intrahepatic MAIT cells significantly decreased after EI (T0: 11.1 (14.4) versus T1: 5.3 (9.3)%; p = 0.0029), in conjunction with significant improvements in fibrosis stage and hepatocyte ballooning. Conclusions: These data demonstrate independent benefits from dietary and exercise intervention and suggest a role for intrahepatic MAIT cells in the observed histological improvements in NAFLD.     

雌孕激素对乳腺癌MCF-7细胞热休克蛋白mRNA水平的影响

目的：研究一定浓度雌、孕激素作用于MCF-7细胞一定时间后,细胞中VEGF、Hsp70、Hsp90和NF-κBp50 mRNA水平上的变化,进而揭示雌、孕激素对乳腺癌MCF-7细胞株作用的分子生物学机制.方法：体外培养人乳腺癌MCF-7细胞株,分4组给予不同处理因素,分别为对照组、雌激素组、孕激素组和雌孕激素混合组.给药72 h后MTT法测细胞570 nm分光光度值.RT-PCR法观察各组Hsp70、Hsp90、VEGF和NF-κBp50 mRNA表达水平.结果：与对照组相比,10^-9mol/L雌激素促进细胞生长作用最明显,10-7mol/L孕激素本身对MCF^-7细胞生长无明显影响,但在雌、孕激素混合组孕激素部分抑制雌激素促细胞增殖作用,具有统计学意义.与对照组相比,雌激素上调细胞中Hsp70、Hsp90、VEGF和NF-κBp50 mRNA水平;孕激素组Hsp70、Hsp90、VEGF和NF-κBp50 mRNA水平与对照组差异无统计学意义.孕激素可以部分抑制雌激素对MCF-7细胞Hsp70、Hsp90、VEGF和NF-κBp50 mRNA的上调作用.结论：雌激素促进乳腺癌细胞生长,可能是雌激素与癌细胞受体结合后通过某种途径刺激Hsp70、Hsp90、NF-κBp50及VEGF的表达,进而抑制癌细胞的凋亡,促进细胞增殖.孕激素通过抑制雌激素对Hsp70、Hsp90、NF-κBp50和 VEGF mRNA的上调作用,进而抑制其促进细胞增殖作用.雌、孕激素促进或抑制细胞增殖,与其对Hsp70、Hsp90、NF-κBp50及VEGF基因的调控是密切相关的.