细胞治疗作为新兴的现代生物医学技术,促进了以精准医学和系统生物学为代表的治疗变革,也不断地与中医关于生命认识的整体观和"天人合一"的思维接近.细胞治疗为肿瘤等多种疾病的治疗提供了新的策略,但其不良反应和应用局限制约了其在临床上的广泛应用.中医药与细胞治疗联合使用,发挥协同作用,在提高细胞治疗疗效、减轻毒副作用方面已经做了一些探索和尝试.概述中医药在免疫细胞治疗、干细胞治疗和基因修饰体细胞治疗中的应用现状,初步探讨中医药联合细胞治疗在未来的发展趋势.

基因编辑技术可针对性增强体细胞的靶向性,一定程度上解决了传统过继性免疫疗法在肿瘤等多种疾病治疗中显露出的靶向性差等问题.最具代表性的是嵌合抗原受体T细胞(CAR-T)免疫疗法,其在血液系统肿瘤治疗中已取得良好疗效,此外,T细胞受体基因工程化的T细胞(TCR-T)、基因修饰的树突状细胞以及基因修饰的干细胞在多种疾病治疗的临床研究中也显现了良好的安全性和有效性.目前国内外多家研究机构和生物公司已在基因修饰体细胞行业加速布局,相关产业即将进入快速发展期,基因修饰体细胞疗法的临床转化应用具有广阔前景,对目前己经开展疾病临床研究的几种基因修饰免疫细胞疗法进行总结.

干细胞研究和临床试验发展迅速,目前全球干细胞临床研究排名前三的国家或地区分别是美国、欧洲和中国,干细胞治疗种类以造血干细胞为主,间充质干细胞日益增长,神经干细胞和多能干细胞的临床试验也相对较多.目前全球己有14款干细胞药物上市,超过一半以上是间充质干细胞治疗产品.中国共有87个干细胞临床项目完成备案,其中间充质干细胞备案项目最多.细胞治疗是按照医疗技术还是药品来监管,世界各国有所不同.在美国按细胞组织类产品风险高低进行归类监管,欧盟以先进技术治疗医学产品归类监管,日本按照再生医学产品管理,中国目前进行机构和项目双备案制度.在严格分类管理的基础上,无论欧盟的医院豁免制度、日本的条件限制性准入政策,还是中国从药品-第三类医疗技术-备案制管理政策的变迁,都为干细胞及其他细胞治疗产品的研究和应用提供了科学而快速发展的政策保障.对国内外干细胞临床研究及应用的发展现状进行综述,同时分析各国干细胞临床研究相关的法律法规与质量控制监管政策.

随着细胞治疗相关研究的不断推进,部分细胞治疗产品已经应用于临床研究.然而,我国相应的细胞治疗监管政策仍需进一步完善,这有利于我国的细胞治疗产业发展,同时保障患者利益.从不同类型的细胞治疗出发,结合现有技术能力,就目前细胞治疗可行的质量控制和管理方法做出论述,以期起到抛砖引玉的作用,引导社会各方制定更为科学规范的管理措施.

随着医学技术的不断进步和科学研究的不断深入,肿瘤免疫细胞治疗作为一种新的治疗方式受到社会各界的广泛关注,成为辅助肿瘤治疗的一种新手段.过继免疫细胞治疗相继出现了不同类型的细胞——T淋巴细胞、细胞因子诱导的杀伤(CIK)细胞、树突状细胞(DC)-CIK细胞、自然杀伤(NK)细胞和肿瘤浸润性淋巴细胞(TIL)细胞等.日前开展的多种类型的免疫细胞临床研究在肿瘤治疗中已经体现出显著的疗效,肿瘤免疫治疗成为快速发展的研究领域.如何选择肿瘤免疫细胞治疗方法和时机,治疗过程中的质量控制和质量管理应当怎么做,如何得到更有说服力的循证医学证据,是值得深入探索的重要问题.对目前的肿瘤免疫细胞治疗现状和未来5年发展做一概述,初步探讨临床研究中的质量控制和质量管理方向.

The prevalence of Candida infection induced by non-albicans Candida (NAC) species is increasing. However, as a common NAC species, C. tropicalis has received much less study in terms of host immunity than C. albicans has. In this study, we evaluated the pro-inflammatory cytokine responses evoked by C. tropicalis and determined whether dectin-1 and downstream NF-κB and mitogen-activated protein kinases (MAPKs) signaling pathways played roles in inflammation in human peripheral blood mononuclear cells (PBMCs) and THP-1 macrophage-like cells. Exposure of PBMCs and THP-1 macrophage-like cells to C. tropicalis led to the enhanced gene expression and secretion of TNF-α and IL-6 in a time- and dose-dependent manner. THP-1 macrophage-like cells being challenged by C. tropicalis resulted in the activation of the NF-κB, p38, and ERK1/2 MAPK signaling pathways. We also found that the expression of dectin-1 was increased with C. tropicalis treatment. These data reveal that dectin-1 may play a role in sensing the inflammation response induced by C. tropicalis and that NF-κB and MAPK are involved in the downstream signaling pathways in macrophages.

Objective To construct a native promoter-regulated Aspergillus fumigatus strain containing red fluorescent protein-labeled calmodulin (CaM-RFP),and to observe the dynamic distribution of calmodulin during the growth of Aspergillus fumigatus.Methods Bilateral flanking sequences of Aspergillus fumigatus calmodulin gene were designed,and plasmids containing the two flanking sequences and mRFP-Aspergillus fumigatus pyrG gene (mRFP-AfpyrG) were amplified separately.The final linear PCR product for transformation was generated from the above three PCR products by fusion PCR.Then,the above linear fragment was transferred into the Aspergillus fumigatus strain by protoplast transformation,so as to construct the CaM-RFP Aspergillus fumigatus strain.The monoclonal colony was picked from the screening medium and subjected to culture.Then,the stablest fluorescent monoxenic strain of Aspergillus fumigatus was selected,and the transformant was verified by PCR.The recombinant strain and wild-type stain were cultured on solid nutrient media separately,and the morphology of these strains was observed by fluorescence microscopy at different time points.Additionally,the above 2 strains were cultured in liquid media separately,and XTT assay was performed to evaluate the growth activity of strains.Microscopy was also conducted to dynamically observe the CaM-RFP Aspergillusfumigatus strain,and analyze the spatial and temporal distribution of calmodulin during the growth and development of Aspergillus fumigatus.Results The fluorescent phenotype and PCR identification results both indicated the successful construction of the CaM-RFP Aspergillus fumigatus strain.The growth activity at 24 hours did not differ between the recombinant strain and wild-type stain (A490:0.689 ± 0.081 vs.0.678 ± 0.054,t =1.32,P ＞0.05),so did the morphology.During the polarized growth of Aspergillus fumigatus,calmodulin was always at the top of the hyphae,germination site of the hyphal branch and the top of new branches.Conclusion Calmodulin may be involved in the regulation of spore germination and polar hyphal growth of Aspergillus fumigatus.

Objective To investigate the roles of Dectin-1 in phagocytosis of Candida albicans (C.albicans) by macrophage-like cells derived from a human acute monocytic leukemia cell line THP-1.Methods THP-1 macrophage-like cells served as the target cells,and were transfected with small interfering RNA (siRNA) targeting Dectin-1 to down-regulate the expression of Dectin-1 receptor (siRNA-Dectin-1 group).THP-1 macrophage-like cells transfected with nonsense siRNA (siRNA-NC) served as a negative control group.After transfection,the THP-1 macrophage-like cells in the above 2 groups were cocultured with heat-killed C.albicans separately.And then,fluorescence microscopy was performed to count THP-1 macrophage-like cells phagocytosing C.albicans,and flow cytometry was used to determine the mean fluorescence intensity (MFI) of dihydrorhodamine (DHR)-123 fluorescent cells.Statistical analysis was done by one-way analysis of variance (ANOVA) and t test with the SPSS19.0 software.Results After transfection with siRNA-Dectin-1,the mRNA and protein expression of Dectin-1 significantly decreased in THP-1 macrophage-like cells (t =26.163,P ＜ 0.001).After 1-,2-,4-hour co-culture of THP-1 macrophagelike cells with C.albicans,fluorescence microscopy showed that the phagocytosis rates of C.albicans by THP -1 macrophage-like cells were significantly lower in the siRNA-Dectin-1 group than in the negative control group (17.5％ vs.22.1％,18.6％ vs.24.3％,39.2％ vs.59.1％,respectively,all P ＜ 0.05),so were the percentage of THP-1 macrophage-like cells phagocytosing more than 3 C.albicans cells (2.2％ vs.4.7％,2.5％ vs.5.4％,5.1％ vs.8.3％,respectively,all P ＜ 0.05).After 30-minute,1-,2-and 4-hour co-culture of THP-1 macrophage-like cells with DHR-123-labelled C.albicans,flow cytometry showed that the MFI of C.albicans-phagocytosing cells was significantly lower in the siRNA-Dectin-1 group than in the negative control group (36.8 vs.45.7,54.3 vs.62.4,72.1 vs.84.9,93.6 vs.116.7,respectively,all P ＜ 0.05).Conclusion Dectin-1 receptor plays an important role in the phagocytosis of C.albicans by macrophages.

Dectin-1 is the critical sensor for β-glucan from Candida which is the most common human fungal pathogen and cause superficial and system infection. MicroRNAs (miRNAs) play crucial roles in regulating innate immunity. However, the functional role of miRNAs in inflammatory response dependent on the activation of dectin-1 pathway has not been defined. In the present study, we found insoluble β-glucan from the cell wall of Candida albicans (CaIG) was able to increase the production of of IL-6 and TNFα through Dectin-1-Syk-NF-κB and p38MAPK pathway. MiRNAs profiles combined with real-time PCR validation revealed that miR-146a, miR-30-5p, miR-210-3p expression level were increased in THP-1 cells treated with CaIG. The interaction between Dectin-1 and CaIG resulted in an long lasting increase of miR-146a expression dependent on Dectin-1-Syk-NF-κB, p38MAPK, contrasting with a rapid and transient increase of IL-6 and TNFα. Overexpression of miR-146a significantly suppressed the production of IL-6 and TNFα. MiR-146a mimics inhibited CaIG-induced activity of p-IκBα and translocation of NF-κB p65. Luciferase reporter assays showed miR-146a inhibited NF-κB promoter-binding activity. Together, our data suggest miR-146a may play the potent negative feedback regulator in inflammatory response following Dectin-1 stimulation.

Objective To evaluate the effect of amphotericin B on the production of tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) and activation of p38 mitogen-activated protein kinases (p38MAPK) in a human acute monocytic leukemia cell line (THP-1).Methods Cultured THP-1 cells were divided into several groups:blank control group receiving no treatment,amphotericin B groups treated with 2,4 and 8 mg/L amphotericin B separately,positive control group treated with 100 μg/L β-glucosan or 100 mg/L lipopolysaccharide.Real-time fluorescence-based quantitative PCR was performed to determine the mRNA expression of TNF-α and IL-8 after the THP-1 cells were treated with different stimuli for some durations.Enzyme-linked immunosorbent assay (ELISA) was conducted to detect the level of TNF-α in the culture supernatant of THP-1 cells after 24-hour treatment with 8 mg/L amphotericin B,and Western blot analysis to measure the levels of p38MAPK and phosphorylated p38MAPK after 30-minute treatment with 8 mg/L amphotericin B.Results After 6-hour treatment with 2,4 and 8 mg/L amphotericin B separately,the mRNA levels of TNF-α in THP-1 cells (7.55 ± 1.17,19.47 ± 2.91,57.22 ± 0.65) and IL-8 (2.98 ± 0.04,5.22 ± 1.35,11.82 ± 1.66) were all significantly higher than those in the blank control group (TNF-α:1.00 ± 0.07,P ＜ 0.01,0.001,0.001 respectively;IL-8:1.01 ± 0.23,P ＜ 0.01,0.001,0.001 respectively).After the treatment with 8 mg/L amphotericin B for 1,3,6 hours,the mRNA levels of TNF-α (8.61 ± 0.30,10.75 ± 0.08,56.98 ± 2.43) and IL-8 (2.63 ± 0.28,5.35 ± 0.98,11.73 ± 1.18) in THP-1 cells were all significantly higher than those in the blank control group (TNF-α:1.18 ± 0.17,P ＜ 0.05,0.01,0.001;IL-8:1.23 ± 0.11,P ＜ 0.05,0.01,0.001).After 24-hour treatment with 8 mg/L amphotericin B,the level of TNF-α in the culture supernatant of THP-1 cells was significantly higher than that in the blank control group (4 039.06 ± 223.87 ng/L vs.96.31 ± 0.26 ng/L,P ＜ 0.001).Conclusion Amphotericin B can promote the p38MAPK phosphorylation and increase the levels of TNF-α and IL-8 in human THP-1 cells in vitro,suggesting its immunomodulatory effects.

Objective To evaluate effects of the yeast form of Sporothrix schenckii on activation of p38 mitogen-activated protein kinase (p38MAPK) and expression of interleukin-6 (IL-6) in macrophagelike THP-1 cells,which were differentiated from the human acute monocytic leukemia cell line THP-1.Methods THP-1 macrophage-like cells were divided into 3 groups to be treated with the yeast form of Sporothrix schenckii at a concentration of 2 × 106 colony-forming units (CFU)/ml (yeast form group),100 mg/L curdlan (curdlan group) and RPMI 1640 medium (blank control group) respectively.Real-time fluorescence-based quantitative PCR was performed to measure the mRNA expression of IL-6 in THP-1 macrophage-like cells in the above 3 groups after 3-and 6-hour treatment separately,and enzyme-linked immunosorbent assay (ELISA) to detect the level of IL-6 in the culture supernatant of THP-1 macrophagelike cells after 24-hour treatment.Western blot analysis was conducted to determine the protein expression of p38MAPK and phosphorylated p38MAPK (p-p38MAPK) in the above 3 groups after 30-and 60-minute treatment separately.Other THP-1 macrophage-like cells were pretreated with 100 nmol/L dexamcthasonc (a p38MAPK inhibitor) for 30 minutes,and then were divided into 3 groups to be treated with the yeast form of Sporothrix schenckii,curdlan and RPMI 1640 medium respectively,and changes in the level of pp38MAPK and mRNA expression of IL-6 were also detected.Statistical analysis was carried out with SPSS19.0 software by using one-way or multi-way analysis of variance and least significant difference (LSD) test.Results Significant differences in the mRNA expression of IL-6 in THP-1 macrophage-like cells were observed among the yeast form group,curdlan group and blank control group (F =5 552.22,P ＜0.001) after 3-hour treatment (56.81 ± 7.36,26.69 ± 1.22 and 0.97 ± 0.05,respectively) and 6-hour treatment (378.03 ± 16.67,276.24 ± 39.13 and 1.02 ± 0.04,respectively).Additionally,the yeast form group showed significantly higher mRNA expression of IL-6 after 6-hour treatment than that after 3-hour treatment (q =16.74,P ＜ 0.001).After 24-hour treatment,the level of IL-6 in the culture supernatant of THP-1 macrophage-like cells also significantly differed among the yeast form group,curdlan group and blank control group (59.96 ± 18.16 pg/L,91.01 ± 17.27 pg/L,5.50 ± 2.30 pg/L,respectively;F =26.62,P ＜ 0.01),and was significantly higher in the yeast form group than in the blank control group (P ＜ 0.01).After 30-and 60-minute treatment,the protein expression of p-p38MAPK was significantly higher in the yeast form group than in the blank control group (both P ＜ 0.01).Moreover,the mRNA expression of IL-6 (4.46 ± 1.03 vs.493.52 ± 113.87,P ＜ 0.001) and protein expression of p-p38MAPK (2.29 ± 0.37 vs.4.55 ±0.46,q =10.81,P ＜ 0.01) were both significantly lower in the yeast form group with dexamethasone pretreatment than in that without dexamethasone pretreatment.Conclusion In vitro treatment with the yeast form of Sporothrix schenckii can enhance the expression of IL-6 in human THP-1 macrophage-like cells by activating the p38MAPK signaling pathway.

Objective:To determine the effects of Sporothrix schenckii yeasts on the activation of NF -κB signal pathway and secretion of TNF-α in human acute monocytic leukemia cells ( THP-1) . Methods: The expression of TNF-αmRNA was detected by Real-time fluorescence quantitative PCR and enzyme-linked im-munosorbent assay respectively. The level of phosphorylated IκBαwas detected by Western blot. NF-κB-p65 nuclear translocation was measured by immunofluorescence. The level of TNF-α mRNA in THP-1 pretreated with 100 nM Dexamethasone ( a NF-κB inhibitor) for 30 minutes was detected by Real-time fluorescence quantitative PCR. Results: The levels of TNF-α mRNA in THP-1 cells treated with Sporothrix schenckii yeasts for 6 hours were increased compared with the blank control group (P<0.001). The secretion level of TNF-α in the Sporothrix schenckii yeasts group was 4610.419±121.501 pg/mL, which was higher than that in the blank group (186.964±98.073 pg/mL), with a significant difference (P<0.001). Phosphorylation IκBαprotein increased obviously and in a time-dependent manner after treated with Sporothrix schenckii yeasts from 30 minutes to 60 minutes. The fluorescent intensity of NF-κB-p65 in the Sporothrix schenckii yeasts group was stronger than that in the blank group. The level of TNF-α mRNA was decreased in the THP-1 macrophages treated with 100 nM dexamethasone in the three groups. Conclusion:Sporothrix schenckii yeasts can increase the expression of TNF-α through enhancing the activation of NF-κB pathway.

Candida parapsilosis is one of the most prevalent Candida species; however, the inflammation response induced by C. parapsilosis and related mechanism received few studies. In this study, we analyzed the pro-inflammatory cytokine responses evoked by C. parapsilosis in human peripheral blood mononuclear cells (PBMCs) and THP-1 cells, determined the signal pathways related to the inflammation response and investigated the expression of dectin-1 modified with C. parapsilosis. Exposure of PBMCs and THP-1 cells to C. parapsilosis led to the increased gene expression and production of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). C. parapsilosis induced TNF-α and IL-6 release in a time- and dose-dependent manner. Western blotting was used to analyze p38, ERK1/2 mitogen-activated protein kinases (MAPKs) and IκB-α phosphorylation and degradation. Nuclear translocation of NF-κB was detected by confocal microscopy. THP-1 cells challenged by C. parapsilosis resulted in the activation of NF-κB and phosphorylation of p38 and ERK1/2 MAPKs. The expression of dectin-1 was up-regulated after the stimulation of C. parapsilosis. Our results suggest that C. parapsilosis could stimulate the inflammatory response, increase the expression of dectin-1 and activate NF-κB and MAPKs signaling pathways in macrophages.