This phase II clinical trial investigated the efficacy and safety of intramuscular injection of plasmid pUDK-HGF, which encodes the human hepatocyte growth factor gene in patients with critical limb ischemia. Resting pain patients (n = 119) and patients with leg ulcers (n = 121) were enrolled as two cohorts and randomized to receive pUDK-HGF treatment on days 0, 14, and 28. In the resting pain cohort, the proportion of patients with complete pain relief on day 180 after receiving pUDK-HGF injection, as the primary outcome, was significantly higher than that of the placebo group on the same day (p = 0.0148). More responders with >50% pain reduction were also observed in the pUDK-HGF groups than in the placebo groups (p = 0.0168). In the ulcer cohort of patients, pUDK-HGF treatment tended to be superior to the placebo in the percentage of patients with both complete ulcer healing and >50% ulcer healing. No significant differences in the incidence of adverse events (AEs) or serious AEs were observed among the groups. The mid-dose pUDK-HGF (6 mg) was the most efficacious, and is therefore an appropriate dose for use in a phase III clinical trial. This study was approved by the China Food and Drug Administration (2013L00637), China Clinical Trial Registry URL: www.chinadrugtrials.org.cn. Unique Identifier: 20130378.

Neorudin(EH)has a good anticoagulant effect.However,the short half-life of EH limits its clinical popu-larization and application.To prolong the half-life of EH,fusion proteins of Fc segment of human IgG and EH were prepared and the anticoagulant activity and pharmacokinetics of the fusion proteins were studied.The fusion genes Fc-EH and Fc-L-EH were cloned into the expression vector pcDNAHC and expressed in CHO cells.The fusion proteins were purified by protein A affinity chromatography and identified by SDS-PAGE and Western Blot.The molecular mass and C-terminal amino acid sequences of the fusion proteins were detected using mass spectrometry.The antico-agulant activities of fusion proteins in vitro and in vivo were detected with clot methods and jugular vein thrombosis rat models,respectively.The half-life of the fusion protein was tested in rats by chemiluminescence immunoas-say.The results of restriction enzyme reaction indicated that the two recombinant expression vectors pcDNAHC-Fc-EH and pcDNAHC-Fc-L-EH were constructed,and the molecular masses of two targeting proteins were measured 68,476,u and 70,542,u,respectively.The correct C-terminal amino acid sequences of the fusion proteins were ob-tained.The anticoagulant activity of the fusion protein Fc-EH is 256 ATU/mg,whereas that of Fc-L-EH is 64 ATU/mg.The results of jugular vein thrombosis rat models indicated that the fusion proteins of Fc-EH dose-dependently inhibited thrombosis.In vivo pharmacokinetic,studies showed that the elimination half-life of Fc-EH spanned 39.4,h.These results demonstrate that the Fc-EH fusion protein prolongs the half-life of EH,paving the way for the further studies of its anticoagulant effects.

OBJECTIVE:Our previous phase I clinical trial has confirmed the safety of Adenovirus carrying Hepatocyte Growth Factor gene (Ad-HGF) by intracoronary administration for treating severe coronary artery disease. This study was performed to evaluate the safety and efficacy of Ad-HGF by percutaneous endocardial injection for treating post-infarct heart failure.METHODS:A total of 30 patients (15 in the experimental group and 15 in the control group) with postinfarct heart failure who were not indicated to revascularization and had received the optimal standardized medication therapy were included in the study. Percutaneous endocardial Ad-HGF gene transfer was injected with a catheter-based intramyocardial delivery system in the experimental group. Safety parameters were measured and compared between baseline and follow-ups in the experimental group. The Mean Difference (MD) of efficacy parameters from baseline to 6-month follow-up was measured in both groups and compared with each other.RESULTS:No one suffered from serious adverse events in the experimental group during the 6-month follow-up. The experimental group revealed significant lower left ventricular end-diastolic dimension (LVDd) (68.5 vs. 65.8 MD: -2.69±1.08, P=0.03) and higher LVEF of both echocardiograph (35.2 vs. 39.3, MD: 4.05±0.86, P=0.0005) and single photon emission computed tomography (27.7 vs. 30.6, MD: 2.9±0.8, P=0.003) in the 6-month follow-up than that in the baseline, but the control group did not (P>0.05). Compared to the control group, the experimental group showed significant improvement ranges of lower LVDd (2.6 vs. -2.69, MD: -5.3±1.4, P=0.0009) and higher echocardiographic LVEF (-2 vs. 4.05, MD: 6.1±1.6, P=0.0008) from baseline to 6-month follow-up.CONCLUSION:Percutaneous endocardial administration of Ad-HGF is safe and potentially efficient in improving LVEF and lowering LVDd of patients with post-infarct heart failure.

Objective To investigate the protective role and underlying mechanism of human dental pulp stem cells (DPSCs)-derived exosomes against lipopolysaccharide ( LPS) induced acute lung injury ( ALI) in pulmonary alveolar macrophage(PAM) cells of rats.Methods DPSCs were cultured in the complete culture medium , and their supernatants at passage 6 were collected after serum-free medium treatment for 24 hours.Exosomes were extracted and purified with ultracentrifugation .Rat PAM NR8383 was cultured in 12-well plate and treated with LPS of 1μg/ml alone or together with exosomes.The supernatants were then collected at 0, 6, 12 and 24 h respectively after treatment .Inflammatory cytokine levels of tumor necrosis factor-α(TNF-α)and interleukins (IL-1βand IL-6) in the supernatant were measured by ELISA assay and the expression and phosphorylation level of MAPK (p44/42), NF-κB and IκBαin cell lysates were detected with Western-blotting.Results Compared with control group , the content of TNF-α,IL-1βand IL-6 increased significantly in LPS group (P<0.05), which indicated that the inflammatory cell model was induced successfully .The levels of TNF-αand IL-1βwere obviously attenuated after a high doses of exosomes treatment (P<0.05), and the expression of IL-6 was markedly suppressed after low and high doses of exosomes treatment (P<0.05), compared with the group of LPS treatment alone.The phosphorylation of NF-κB, IκBαand p44/42 was significantly inhibited after treatment with the DPSCs-derived exosomes.Conclusion DPSCs-derived exosomes may have a potential protective effect on LPS-induced ALI, and the underlying mechanism is that the activity of MAPK (p44/42) and NF-κB/IκBαpathways are eliminated by DPSCs-derived exosomes.

在精准医学全面启动的新趋势下,以干细胞和免疫细胞治疗为代表的新型细胞治疗技术也从“个体化”向“精准化”升级和转变.干细胞及其分化来源的细胞成为指导疾病精准治疗的理想模型;嵌合抗原受体T细胞免疫疗法(CAR-T)技术的成功代表着精准免疫细胞治疗时代的到来;肿瘤干细胞为肿瘤的精准治疗提供了更加可靠的靶标和模型;干细胞结合基因修饰和基因编辑等基因治疗技术成为组织损伤修复和疾病治疗的新策略.基因技术已经助力细胞治疗进入精准时代.

BackgroundNeuropathic pain (NP) is a refractory disease in the clinic with a tremendous impact on the quality of life of patients. Gene therapy is a potential strategy for the management of NP. In the present study, we examined the analgesic effect and mechanism of hepatocyte growth factor (HGF) in vitro and in vivo. MethodsWe examined the proinflammatroy gene changes in lipopolysaccharide (LPS)-induced microglia BV2 cells with a quantitative real-time polymerase chain reaction of interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)- and inducible nitric oxide synthase (iNOS). Mechanical stimulation tests were performed five times at 5-min intervals to assess pain thresholds using Von Frey Hair in mice following spared nerve injury (SNI). The glial cell activation of spinal cord was examined by western blotting. Statistical significance was determined by a Tukey's test and a paired t-test. ResultsWe found that recombinant human HGF protein suppressed LPS-induced BV2 cell activation in vitro, marked by the down-regulation of IL-1, IL-6, TNF- and iNOS expression, as well as decrease of nitric oxide production. Moreover, intrathecal injection of naked plasmid encoding HGF gene (pUDK-HGF) significantly attenuated SNI-induced pain behaviors in mice by direct inhibition of spinal cord microglia and astrocyte activation. ConclusionsThe results of the present study indicate that pUDK-HGF can reduce cytotoxicity products released from activated glial cells, which may provide a promising therapeutic strategy for treating NP.

The innovative medical technology industry of regenerative medicine has shown huge economic potential and development prospects both at home and abroad in the last decade. After describing the current situation of industrial development in innovative technology in cell-technology-related regenerative medicine, we investigate, sort, and analyze related policies and regulations, management systems, and existing problems in the United States, the European Union, Japan, Australia, and other developed countries. On this basis, we study the current development situation, governmental regulation policies, and management mechanism of this industry in China. Next, we analyze and summarize the industry development trend, characteristics, and existing problems. Finally, based on experts’ advice and opinions on breaking through the bottleneck of industrial development, we suggest that it is imperative for us to strive to develop the innovative technology industry of cell-technology-related regenerative medicine in China.

Objective To develop a practical method for the determination of recombinant neorudin (referred to as the EH) in yeast fermentation supematants by HPLC,in order to monitor of the relationship between the expression of recombinant EH and induction time during the fermentation process to optimize the induction time for maximizing the yield of EH.Method The relationship between the EH and ultraviolet absorption was detected by HPLC to establish a method to determine the quantity of EH protein.The feasibility of the method was further validated with regard to the sensitivity,precision and recovery rate.The stability of EH and the trace EH in yeast fermentation supernatants were examined using the method.Results A strong absorption peak at 214 nm was found for EH,having high sensitivity with RSD value of 1.4227％.Good linearity in the EH concentration range of 0.012-4.8 mg/ml with r2 =0.9995 was observed.Standard addition recovery rate was between 95％ and 98％.The result of the EH stability showed that the sample had a good preservation stability,with main peak area percentage being over 95％ at 4 ℃ for 24 h or at 20 ℃ for 8 h.The expression of EH in yeast fermentation supematants was related to the induction time of methanol.Conclusion The method for detecting the contents of EH by HPLC is established.Real-time monitoring of EH in yeast fermentation supematants to get the maximum production of EH will be achieved.

Objective To investigate the therapeutic effect of interleukin-2(IL-2)on experimental autoimmune encephalomyelitis(EAE)mice.Methods After establishment of the EAE(experimental autoimmune encephalomyelitis) mouse models with MOG35-55 polypeptides,the mice were grouped according to the neurological function score and divided into control group,EAE group and low dose IL-2 treatment group.A double blind method was used to evaluate the neuro-logical impairment in mice.On the 29th day,pathological experiments were carried out in the mice's brain and spinal cord, hematoxylin-eosin staining was used to evaluate the scoring of inflammatory cell infiltration and luxol fast blue staining was used to evaluate the scoring of demyelinating.The proportion of regulatory T cells(Treg)and NK cells(natural killer cell, NK)was detected by flow cytometry,and the immunohistochemical method was used to detect the expressions of glial fibril -lary acidic protein(GFAP)and myelin basic protein(MBP)in the spinal cord.Results Compared with the EAE group, the neurological function score, the inflammatory cell infiltration score and the demyelinating score of the low dose IL-2 treatment group were reduced.The proportion of Treg cells in the low dose IL-2 treatment group was significantly higher than that in the EAE group,and the proportion of NK cells in the low dose IL-2 treatment group was slightly higher than that in the EAE group The expression of GFAP and MBP was detected by immunohistochemistry.The expression level of GFAP in low dose IL-2 treatment group was significantly lower than that in the EAE group,while the expression level of MBP was higher than that in the EAE group.Conclusion Low dose IL-2 has significant therapeutic effect on EAE mice.

OBJECTIVE To explore the antitumor effects of combined tanshinoneⅠ(TanⅠ),metformin (Met) and aspirin (Asp) on malignant melanoma in mice and the possible mechanisms. METHODS C57BL/6 mice were injected with 0.1 mL B16F10 cells(2.8×109L-1)to establish the subcutaneous trans-plantation tumor model at the right forelimbs axillary.Then,the mice were divided into 8 groups according to body mass,including model group, TanⅠgroup(20 mg·kg-1,ip),Asp group(210 mg·kg-1,orally in drinking water), Met group (70 mg·kg-1, orally in drinking water), Asp+Met group, TanⅠ+Asp group, TanⅠ+Met group and TanⅠ+Asp+Met group,10 mice in each group.Each mouse drank about 7 mL of water every day for a total of 18 d.The mouse body mass was measured every other day and the tumor diameter was calculated every day. The mice were sacrificed after treatment, the tumor mass was measured and the tumor inhibitory rates were counted. The histopathological changes of the liver and spleen were observed with HE staining. The percentage of lymphocytes in the tumor tissue such as CD8+T,CD4+T and Treg cells was detected by flow cytometry.Inflammatory factors such as interleukin-6 (IL-6),IL-1β and tumor necrosis factor-α (TNF-α) were detected by ELISA. RESULTS The body mass (including tumor mass)of mice in different groups increased during the experiment,but that of TanⅠ+Asp+Met group increased more slower than in model group(P<0.01).At the end of the experiment,no lesions were seen in any liver or spleen tissue by pathological observation,and the number of survivors was 8/10(model group),8/10(TanⅠgroup),7/10(Asp group),7/10(Met group),8/10(TanⅠ+Asp group), 8/10 (TanⅠ+Met group), 7/10 (Asp+Met group) and 5/10 (TanⅠ+Asp+ Met group), respectively. Compared with model group,there were no obvious changes in tumor volume or tumor mass in TanⅠ, Asp and Met groups and other two-two joint groups,but the tumor volume and tumor mass in TanⅠ+Asp+ Met group were significantly decreased (P<0.01, P<0.05), and the tumor inhibitory rate in this group was 46.2%.Compared with the model group,the percentage of CD8+T cells increased(P<0.05) in TanⅠ+Asp+Met group,but there were no significant changes in other groups.The contents of IL-6, IL-1β and TNF-α in tumor tissue of TanⅠ+Met group were much higher than in model group(P<0.01, P<0.05,P<0.05)and the content of IL-6 increased in TanⅠ+Asp+Met group(P<0.01).CONCLUSION Combination of TanⅠ,Asp and Met can effectively inhibit the growth of melanoma in mice,which may be related to the increasing percentage of CD8+T lymphocytes and IL-6 in tumor tissue.However there are possibly some side effects.

Clinical trials testing the effects of a single injection of adenovirus carrying the human hepatocyte growth factor gene (Ad-HGF) in patients with chronic ischemic heart failure failed to show consistent improvements in cardiac function. The aim of this study was to evaluate the efficacy of repeated injections of AdHGF in a rat model of postinfarct heart failure. Ad-HGF or Ad-green fluorescent protein (GFP) was administered to Sprague Dawley rat models of postinfarct heart failure via single or fractional repeated intrapericardial injection. Heart function was monitored by magnetic resonance imaging for 4 and 8 weeks after injections. The expression of HGF or factor VIII/Ki-67 was evaluated by Western blot assay or immunofluorescence. We found that Ad-HGF gene expression could be prolonged in vivo by repeated injections and that cardiac function was significantly improved in the Ad-HGF repeat-injection group compared with the Ad-HGF single-injection group. Newly formed capillary density was similarly higher in the Ad-HGF repeat-injection group compared with that in the Ad-HGF single-injection group. We therefore conclude that fractional repeated injections of Ad-HGF may represent a promising therapeutic strategy to improve cardiac function in the setting of postinfarct heart failure.

为建立可产业化的大肠杆菌表达的重组新蛭素(neorudin,EH)生产工艺,主要采取从低密度到高密度发酵的优化思路,优化了发酵罐培养基和诱导时间;比较了超声破碎和反复冻融的目的蛋白提取方法;纯化工艺采用离子交换层析2步法进行:SP Sepharose Fast Flow阳离子交换层析和Source 15Q阴离子交换层析;所得产品用免疫印迹法鉴定,经HPLC检测纯度,凝块法检测抗凝比活性;最后对纯化产品进行结构确证,包括高分辨率质谱、C-末端测序、N-末端测序、二硫键分析.结果显示:优化后的发酵工艺确定基于TB培养基的高密度发酵工艺,菌体OD600可达40左右,每升菌体湿质量可达70 g左右,目的蛋白表达量约400 mg/L;菌体反复冻融离心上清经过纯化获得的目的蛋白HPLC纯度均大于97％,纯化收率为26.12％,抗凝比活性为1 024 ATU/mg,用高分辨率质谱检测其分子质量约为7 415.188 0 ku,N-末端、C-末端和二硫键均与理论相符.建立了EH在大肠杆菌中的高密度发酵和纯化工艺.

Inflammatory pain and neuropathic pain are major clinical health issues that represent considerable social and economic burden worldwide. In the present study, we investigated the anti-nociceptive efficacy of delivery of human proenkephalin gene by a plasmid DNA vector (pVAX1-PENK) on complete Freund's adjuvant (CFA) induced inflammatory pain and spared nerve injury (SNI) induced neuropathic pain in mice. Mice were intramuscularly or intrathecally administered pVAX1 or pVAX1-PENK, respectively. Pain thresholds in the pVAX1-PENK treated mice were significantly higher at day 3, then reached a peak at day 7 and lasted until day 28 after gene transfer, and the analgesic effect of pVAX1-PENK was blocked with naloxone hydrochloride. In contrast, pVAX1 treated mice did not significantly improve pain thresholds. These results indicate that peripheral or spinal delivery of a plasmid encoding human proenkephalin gene provides a potential therapeutic strategy for inflammatory pain and neuropathic pain.

The demand of a plasmid encoding human hepatocyte growth factor gene (pUDK-HGF) in large quantities at high purity and concentration has increased for gene therapy of critical limb ischemia (CLI) in clinical trials. In this article, we produced pUDK-HGF in compliance with current good manufacturing practices at gram scale. The process included a 50-L batch fermentation, continuous alkaline lysis, and integrated three-step chromatography on Sepharose 6 Fast Flow, PlasmidSelect Xtra, and Source 15Q. The production process has been scaled up to yield 4.24 ± 0.41 g of pharmaceutical pUDK-HGF from 1.0 kg bacterial cell paste and the overall yield reached range from 58.37 to 66.70%. The final pUDK-HGF product exhibited high purity with supercoiled percentage of > 95.8% and undetectable residual RNA, contaminated protein, and bacterial endotoxin. The phase I clinical study indicates that intramuscular injection of pUDK-HGF is safe, well tolerated, and may provide symptomatic relief to CLI patients. These results show that our manufacturing process of pUDK-HGF is efficient in producing pharmaceutical-grade plasmid DNA and is safe for clinical applications.

We investigated the antinociceptive effect of local intramuscular injection of a plasmid encoding human proenkephalin (pVAX1-hPPE) on postoperative pain in rats. Male Sprague-Dawley rats with incision-induced pain were intramuscularly injected into injured plantaris muscle with empty vector (pVAX1) or pVAX1-hPPE, respectively. Paw mechanical threshold and thermal latency in the 200μg pVAX1-hPPE treated rats were significantly higher at 6h and on 1day, and lasted until day 7 after intramuscular administration, respectively. The analgesic effects were reversed by methylnaltrexone, suggesting that the antinociceptive effect of pVAX1-hPPE was mediated through peripheral opioid receptor pathway. In contrast, incisional or pVAX1-treated rats did not significantly affect pain thresholds. These results demonstrated that single intramuscular injection of pVAX1-hPPE attenuated incision-induced pain in rats, and it is worthy of further study as a potential gene therapy for postoperative pain.

生物治疗作为外科手术、药物治疗、放射治疗之后的第四大治疗技术，已经得到全球的广泛认同。生物治疗主要由细胞治疗、基因治疗、组织工程等领域组成，其中细胞治疗由于发展的快速性和重要性，引起了科学界与临床应用的更多关注。作为一个冉冉升起的新兴医药产品领域，细胞治疗受到了全球发达国家政府的战略重视和投入，也成为产品研发和风险投资关注的热点领域。目前以干细胞治疗和免疫细胞治疗为主流的细胞治疗产品在人类疾病治疗中的地位和价值已经初步显现，尤其是在众多未满足需求的难治性疾病领域，如恶性肿瘤、疑难重症的治疗和严重创伤修复的再生医学领域，细胞治疗已经成为弥补传统治疗不可或缺的有效手段。对于一些传统药物或治疗手段束手无策的重大疾病，如重症肝病、移植物抗宿主病（GvHD）、肾移植排斥、系统性红斑狼疮、帕金森病、老年痴呆等，细胞治疗均显示出了明确的疗效。

Objective To compare two sources of mesenchymal stem cells ( MSCs) from human placenta and umbilical cord, and to optimize a technical solution for bench or clinical studies of MSCs.Methods MSCs were isolated from human placenta and umbilical cord and expanded for analysis.The cell morphology was observed under invert microscope, the immunophenotypic feature of MSCs was analyzed with flow cytometer, the cell proliferation ability was determined by cell cycle assay and cell doubling time, the cell differentiation potential was evaluated by osteogenic and adipogenic induction in vitro as well.Results Both sources of MSCs were adherent cells and exhibited fusiform and fibrous morphology. Furthermore, both MSCs high expressed CD90 and CD105, and were negative for the markers of CD34, CD45 and HLA-DR.The population doubling time of MSCs form human placenta and umbilical cord was 39.5 h and 40.8 h separately, and the results of cell cycle analysis showed that the percent of the two sources of MSCs in G0/G1 phase was 52.12%and 57.50% respectively. The above results demonstrated that both sources of MSCs possessed the similar biological characteristics in morphology, phenotype and as well as proliferation ability.In addition, both of them could be induced into osteoblasts and adipocytes in vitro.Conclusion MSCs from human placenta have the similar biological characteristics to these from human umbilical cord, and both of them are better candidates for bench and clinical research.

BACKGROUND:Placenta is a valuable source of mesenchymal stem cels for stem cel therapy and future application in the field of regenerative medicine. However, conventional methods cannot acquire a large amount of purified human placenta-derived mesenchymal stem cels. Here, we present a new method for isolating human placenta-derived mesenchymal stem cels suitable for banking strategies and for future clinical applications. OBJECTIVE:To analyze the biological characteristics of human placenta-derived mesenchymal stem cels cultured by tissue dissociating and colagenase digestion. METHODS: Human placenta-derived mesenchymal stem cels were obtained from human placenta by tissue dissociating and colagenase digestion method. Immunophenotype was analyzed by flow cytometry. Growth curve was determined by MTT assay, and differentiation ability was evaluated byin vitro adipogenic, osteogenic and chondrogenic induction as wel. RESULTS AND CONCLUSION:Human placenta-derived mesenchymal stem cels could be passaged stablyin vitro. Furthermore, the cels expressed CD73, CD90, CD105, but were negative for the markers of CD11b, CD19, CD34, CD45, and HLA-DR. Human placenta-derived mesenchymal stem cels proliferated actively and began to grow logarithmicaly at days 3-5 folowed by a plateau period at day 6. In addition, the isolated cels could be induced into adipocytes, osteocytes, chondrocytesin vitro. In a word, the results of this study demonstrated that the tissue dissociating and colagenase digestion method is an efficient method for obtaining a large amount of human placenta-derived mesenchymal stem cels that can be stably cultured in vitro and have strong proliferative ability.

Objective To establish an aging model of mesenchymal stem cells (MSCs) and to investigate aging related biological mechanism for the purpose of studying the senesence of MSCs .Methods MSCs were separated and purified from human placenta, and the cells of the third passage(P3-MSCs) were cultured in the medium for 2 hours, then 100,200 and 300 μmol/L hydrogen peroxide ( H2 O2 ) was added to the cells for 2 hours to establish the MSCs aging model in vitro. Biological characteristics of aging MSCs were evaluated by cell cycle assay and senescence associated β-galactosidase staining.The expression of p16,p21 and p53 genes was further measured using quantitative real-time PCR (RT-PCR).Re-sults Compared with the control , the number of MSCs treated with 200μmol/L H2 O2 for 2 hours was significantly decreased and the cells displayed less adipogenic ,osteogenic and chondrogenic differentiation .Moreover ,after exposure to 200 μmol/L H2 O2 , the majority of the cells were in the G 0/G1 phase as showed by cell cycle analysis .The percentage of senescence-associated β-galactosidase-positive cells was increased , and the expression of p 16 , p21 and p53 mRNA and protein was significantly increased.Conclusion The results of this study has demonstrated that the H 2 O2 (200 μmol/L) can be used to establish the aging model of MSCs in vitro, and the cellular phenotypic alteration may attribute to the cell cycle associated gene expression (p16, p21, and p53).