自体富血小板血浆局部注射治疗糖尿病足溃疡的临床研究

目的探讨局部注射自体富血小板血浆（platelet-rich plasma，PRP）治疗糖尿病足溃疡的疗效。方法将 2017 年 10 月—2018 年 10 月收治且符合选择标准的 90 例糖尿病足溃疡患者随机分为 3 组：PRP 注射组（A 组，创面及创周局部注射 PRP+水凝胶敷料覆盖创面）、PRP 覆盖组（B 组，PRP 凝胶+水凝胶敷料覆盖创面）和对照组（C 组，单纯水凝胶敷料覆盖创面），每组 30 例。3 组患者性别、年龄、侧别、病程及术前糖化血红蛋白、创面面积、Wagner 分级等一般资料比较，差异均无统计学意义（P>0.05），具有可比性。比较 3 组患者治疗次数、住院时间及 A、B 组 PRP 使用总量。治疗期间观察创面愈合情况，首次清创术后 3 个月测算创面愈合率。 结果A、B、C 组治疗次数分别为（10.2±0.8）、（11.4±0.6）、（12.5±0.5）次，A、B 组 PRP 使用总量分别为（306±24）、（342±18）mL，组间比较差异均无统计学意义（P>0.05）。A、B、C 组住院时间分别为（40.5±1.8）、（62.1±2.3）、（88.6±1.4）d，差异有统计学意义（P<0.05）。治疗过程中，3 组创面坏死渗出均逐渐减少，创面面积逐渐缩小；A 组均明显优于 B、C 组，B 组优于 C 组。首次清创术后 3 个月，A、B、C 组创面愈合率分别为 93.2%±0.8%、52.1%±1.1%、21.3%±1.3%，组间比较差异均有统计学意义（P<0.05）。 结论PRP 能有效促进糖尿病足溃疡创面修复，而且 PRP 局部注射疗效优于 PRP 凝胶覆盖。     

Anastatica hierochuntica (L.) methanolic and aqueous extracts exert antiproliferative effects through the induction of apoptosis in MCF-7 breast cancer cells

Breast cancer therapy using anticancer bioactive compounds derived from natural products as adjuvant treatment has gained recognition due to expensive and toxic conventional chemotherapeutic drugs. The whole plant of Anastatica hierochuntica (L.) (A. hierochuntica) has been investigated for its pharmacologically important anticancer properties but without categorizing the biological activities of the plant parts. We assessed the anticancer potential of different parts of A. hierochuntica (seeds, stems and leaves) and explored their mechanisms of action using the human breast cancer cell line, MCF-7. Currently, we investigated the antiproliferative effects of methanolic (MSD, MST, ML) and aqueous (ASD, AST, AL) extracts of A. hierochuntica plant parts on the MCF-7 cells using cell viability assays. Flow cytometry, Western Blot, DNA fragmentation, and gene expression assays were performed to evaluate apoptosis and cell cycle regulatory proteins. The results indicate that the methanolic and aqueous extracts decreased MCF-7 cell viability in a dose-dependent manner. The induction of apoptosis was observed in all the methanolic and aqueous-treated MCF-7 cells. The cell death process was confirmed by the visualization of DNA fragmentation and cleavage of the intrinsic apoptotic pathways, caspase-9 and caspase-3, the key enzyme causing apoptosis hallmarks. In addition, the most pro-apoptotic extracts, ASD and ML, up-regulated the expression of pro-apoptotic Bax, tumor suppressor TP53 genes and the cyclin inhibitor CDKN1A gene. In conclusion, of the aqueous and methanolic extracts of A. hierochuntica plant parts exerting antiproliferative effects through the induction of apoptosis in breast cancer MCF-7 cells, ASD and ML extracts were the most promising natural-based drugs for the treatment of breast cancer.     

永生化人肝星状细胞株WM07的建立

目的 以慢病毒载体介导猿猴病毒40（simian virus 40，SV40）大肿瘤抗原（large tumor antigen，TAg）转染原代人肝星状细胞（hepatic stellate cell，HSC）获得永生化HSC株。方法 以含SV40 TAg cDNA序列的SV40 tsA58质粒为模板，PCR获取全长SV40 TAg序列，经TOPO克隆进入pENTR^TMTOPO^®；载体，PCR鉴定获得pENTR-SV40 TAg Entrez质粒，再经LR重组反应将SV40 TAg cDNA克隆到目标慢病毒载体pLenti4/V5-DEST，PCR鉴定pLenti4/V5-GW/SV40 TAg质粒，进一步用该质粒转染工程细胞293FT进行假病毒包装，用含包装病毒的细胞培养上清液转染原代人HSC，经博来霉素筛选获得稳定表达SV40 TAg的人HSC株（命名为WM07），并对细胞进行SV40 TAg及α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）表达的检测和鉴定。结果 对pENTR-SV40 TAg Entrez质粒及pLenti4/V5-GW/SV40 TAg慢病毒表达质粒进行PCR测序，结果证实所构建的质粒含ATG转录起始位点及SV40 TAg cDNA序列。对经过转染、筛选获得的WM07行免疫荧光染色，结果显示SV40 TAg在细胞核表达和定位，细胞质内均表达活化的HSC标志物α-SMA。结论 pLenti4/V5-GW/SV40 TAg质粒构建成功。经质粒转染，获得永生化HSC株WM07，可稳定传代并用于肝纤维化研究。     

Cancer combinatorial immunotherapy using anti-OX40 agonist and anti-PD-L1 antagonist: a patent evaluation of US2018256711A1

Introduction: OX40 is checkpoint inhibitor in cancer that coordinates the downregulation of the proliferation of antigen-specific lymphocytes. There is a great need to discover and develop new therapies focused on inhibiting the action of OX40 and consequently improving the immune response in the various types of cancer. Authors of patent US2018256711A1 propose a method to eradicate cancer that utilizes anti-OX40 agonist antibody in combination with anti-PD-L1 antagonist antibody.

Areas covered: Patent US2018256711A1 describes a method of cancer combinatorial treatment consisting of the utilization of a pharmaceutical cocktail containing anti-OX40 and an anti-PD-L1 antibody.

Expert opinion: The results of the clinical trials only support trials regarding the tolerability of combinatorial therapy, even when the objectives of determining the safety and pharmacokinetics of the treatment are proposed.     

Effect of curcumin on permeability of coronary artery and expression of related proteins in rat coronary atherosclerosis heart disease model

Objective: Our objective is to explore the effect of curcumin on permeability of coronary artery and expression of related proteins in rat coronary atherosclerosis heart disease model. Methods: 45 healthy male Wistar rats of clean grade were selected and divided into treatment group, model control group and blank control group. The rats in the treatment group and model control group received high-fat diet for 12 weeks and intraperitoneal injection of VD₃ to establish rat coronary atherosclerosis heart disease model. After modeling, the rats in the treatment group received gavage of 100 mg/(kg·d) curcimin, and the rats in the model control group and blank control group received gavage of 5 ml/(kg·d) distilled water, the intervention time was 4 weeks. After intervention, the rats were killed, and the hearts were dissected to obtain the samples of coronary artery. After embedding and frozen section, immunofluorescence method was used to detect the change of endarterium permeability in 3 groups, Western blot was used to detect matrix metalloproteinase-9 (MMP-9) and CD40L in coronary artery tissue, and enzyme linked immunosorbent assay (ELISA) was used to detect serum tumor necrosis factor-α (TNF-α) and C reaction protein (CRP). Results: After modeling, compared with the blank control group, total cholesterol (TC), triglyceride (TG) and low density lipoprotein cholesterin (LDL-c) in the treatment group and model control group were significantly higher (P<0.05), however, high density lipoprotein cholesterin (HDL-c) was significantly lower. The pathological sections showed that there was lipidosis in rat coronary artery in treatment group and model control group, indicating that the modeling was successful. Immunofluorescence showed that there was only a little fluorochrome permeability in artery in blank control group, there was some fluorochrome permeability in artery in the treatment group and there was a lot of fluorochrome permeability in artery in the model control group. MMP-9 and CD40L in coronary artery tissue in the model control group were significantly higher than the treatment group (P<0.05), MMP-9 and CD40L in coronary artery tissue in the treatment group were significantly higher than the blank control group (P<0.05); serum TNF-α and CRP in the model control group were significantly higher than the treatment group (P<0.05), which were significantly higher in the treatment group than the blank control group (P<0.05). Conclusion: Rat coronary atherosclerosis heart disease model can be successfully established by feeding with high-fat diet and intraperitoneal injection of VD₃, the permeability of coronary artery in coronary heart disease rat model is significantly increased, which may be related to up-regulation of MMP-9, CD40L, TNF-α and CRP expression. Application of curcumin can inhibit expression of MMP-9, CD40L, TNF-α and CRP to improve the permeability of coronary artery.     

Different roles of soluble CD40 ligand in central nervous system damage

ABSTRACT

Backgroud and purpose: Soluble CD40 ligand (sCD40L) plays an important role in inflammation and autoimmune disorders. There is still a controversy regarding sCD40L in neuromyelitis optica spectrum disorders (NMOSD) and multiple sclerosis (MS). Herein the aims of this study were to evaluate the levels of sCD40L in patients with NMOSD, MS, and other noninflammatory neurological diseases; to investigate its potential relationship with laboratory parameters, glial fibrillary acidic protein (GFAP), thrombopoietin (TPO) and IL-6; and to address whether serum sCD40L levels in acute attacks of NMOSD patients were decreased after treatment with immunoglobulins, plasma exchange, or methylprednisolone.Materials and methods: We enrolled 13 patients with NMOSD, 9 patients with MS, and 9 patients with other noninflammatory neurological diseases. The levels of sCD40L, IL-6 were measured by cytokine multiplex assay. GFAP levels were measured by ELISA.Results: Both serum and cerebrospinal fluid (CSF) sCD40L levels were increased in NMOSD and MS. No differences were found in serum and CSF sCD40L levels between NMOSD and MS. The CSF sCD40L levels were positively correlated with the CSF cell counts in NMOSD, whereas serum sCD40L levels were positively correlated with the albumin index in MS. Furthermore, the levels of CSF sCD40L were positively correlated with CSF GFAP levels in NMOSD. Serum sCD40L levels were correlated with serum TPO levels in MS. No correlation was found between levels of sCD40L and IL-6 in NMOSD and MS. No statistically meaningful difference between NMOSD patients with or without immunotherapy. Conclusions: Our study suggests that sCD40L can contribute to the destruction of the blood-brain barrier in MS, whereas it may contribute to CNS in?ammation in NMOSD. The serum sCD40L concentrations were not changed after treatment with immunoglobulins, plasma exchange, or methylprednisolone in acute attacks of NMOSD.     

TLR9联合CD40信号对ITP患儿外周血中IL10+ CD3-细胞亚群改变的研究

目的：探讨TLR9联合CD40信号对儿童免疫性血小板减少症（ITP）患儿外周血单个核细胞（PBMC）中CD3-细胞表达IL-10的作用及影响。方法：采用流式多因子阵列检测技术，分析8例ITP患儿和7例正常对照血浆样本中IL-10含量；分离8例ITP患儿及7例正常对照外周血PBMC，并经CpG联合CD40L体外刺激培养5 h， 采用流式胞内外染色及检测技术，比较ITP患儿与正常对照组PBMC中CD3-和CD3+亚群比例、IL-10表达能力及细胞存活率的差异。结果：ITP患儿血浆中IL-10含量显著高于正常对照组（P＜0.05）；CpG联合CD40L刺激后的ITP患儿和正常对照组PBMC中CD3-细胞比例与未刺激组相比均显著升高，而CD3+ T淋巴细胞比例显著下降（P＜0.05）；ITP患儿刺激组中CD3- IL-10+细胞的比例显著高于正常对照刺激组（P＜0.05）；与正常对照组相比，未刺激组ITP患儿CD3-细胞存活率显著下降（P＜0.05）。结论：TLR9联合CD40信号的活化能导致ITP患儿外周血IL10+ CD3-细胞比例升高，细胞死亡率升高，可能与ITP疾病进展有重要关系。