New model for cardiomyocyte sheet transplantation using a virus-cell fusion technique

AIM: To facilitate close contacts between transplanted cardiomyocytes and host skeletal muscle using cell fusion mediated by hemagglutinating virus of Japan envelope(HVJ-E) and tissue maceration. METHODS: Cardiomyocytes(1.5 × 106) from fetal rats were first cultured. After proliferation, some cells were used for fusion with adult muscle fibers using HVJ-E. Other cells were used to create cardiomyocyte sheets(area: about 3.5 cm2 including 2.1 × 106 cells), which were then treated with Nile blue, separated, and transplanted between the latissimus dorsi and intercostal muscles of adult rats with four combinations of HVJ-E and/or Na OH maceration: G1: HVJ-E(+), Na OH(+), Cardiomyocytes(+); G2: HVJ-E(-), NaO H(+), Cardiomyocytes(+); G3: HVJ-E(+),Na OH(-), Cardiomyocytes(+); G4: HVJ-E(-), Na OH(-), Cardiomyocytes(-). At 1 and 2 wk after transplantation, the four groups were compared by detection of beating domains, motion images using moving target analysis software, action potentials, gene expression of MLC-2v and Mesp1 by reverse transcription-polymerase chain reaction, hematoxylin-eosin staining, and immunostaining for cardiac troponin and skeletal myosin.RESULTS: In vitro cardiomyocytes were fused with skeletal muscle fibers using HVJ-E. Cardiomyocyte sheets remained in the primary transplanted sites for 2 wk. Although beating domains were detected in G1, G2, and G3 rats, G1 rats prevailed in the number, size, motion image amplitudes, and action potential compared with G2 and G3 rats. Close contacts were only found in G1 rats. At 1 wk after transplantation, the cardiomyocyte sheets showed adhesion at various points to the myoblast layer in the latissimus dorsi muscle. At 2 wk after transplantation, close contacts were seen over a broad area. Part of the skeletal muscle sarcoplasma seemed to project into the myocardiocyte plasma and some nuclei appeared to share both sarcoplasmas.CONCLUSION: The present results show that close contacts were acquired and facilitated the beating function, thereby providing a new cellular transplantation method using HVJ-E and NaO H maceration.

Highly Selective Photodynamic Therapy with a Short Drug-Light Interval Using a Cytotoxic Photosensitizer Porphyrus Envelope for Drug-Resistant Prostate Cancer Cells

Background: Photodynamic therapy (PDT) is a less invasive cancer treatment using photochemical reactions induced by light irradiation to a photosensitizer (PS). Highly selective PDT with fast accumulation of the PS in target site might be a promising treatment option for drug-resistant prostate cancer facing high incidence rate of elderly men who have no effective treatment options and require a minimally invasive treatment. Hemagglutinating virus of Japan envelope (HVJ-E) allows selective and fast drug delivery to the drug-resistant prostate cancer cells via rapid cell membrane fusion. PS named porphyrus envelope (PE) has been developed by insertion of lipidated protoporphyrin IX (PpIX lipid) into HVJ-E. In this study, we investigated the optimal conditions for PE preparation and laser irradiation for highly selective PDT using PE with a short drug-light interval. Materials and Methods: Human hormon refractory prostate cancer cell line PC-3 and human normal prostate epithelial cell line PNT2 were cultured. PpIX lipid uptake and cytotoxicity of PDT in the cells incubated with PE for 10 min were evaluated by measuring fluorescence intensity and by using a cell counting reagent 24 h after PDT, respectively. Results: PpIX lipid uptake and cytotoxicity of PDT were increased with PpIX lipid concentration. Cytotoxicity of PDT using PE was more than 9 times as strong as that with PpIX lipid and PpIX induced by 5-aminolevulinic acid. Much stronger cytotoxicity was induced in PC-3 cells than PNT2 cells with the ratio of cell death rate for cancer to normal cells up to 4.64 ± 0.09. Conclusions: Fast PS delivery with HVJ-E allows highly selective PDT with a short drug-light interval. Therefore, PDT using PE has a potential to shorten treatment period and reduce side effects of PDT.

A Novel Therapeutic Strategy Combining Use of Intracellular Magnetic Nanoparticles under an Alternating Magnetic Field and Bleomycin

Purpose: The purpose of this study was to present a novel therapeutic strategy combining use of intracellular magnetic nanoparticles (MNPs) under an alternating magnetic field (AMF) and bleomycin (BLM), and to evaluate its therapeutic effect using tumor-bearing mice. Materials and Methods: MNPs (Resovist?, 1.05 mg iron) were incorporated into the hemagglutinating virus of Japan-envelope (HVJ-E) vector (~5 × 109 particles) (HVJ-E/MNPs) by centrifugation at 10,000 × g for 5 min at 4°C. Tumor-bearing mice were prepared by inoculating Colon-26 cells subcutaneously into the backs of BALB/c mice. When the tumor volume reached ~100 mm3, HVJ-E/MNPs and/or BLM were injected directly into the tumor. The AMF was applied to the mice one hour after the injection of agents (AMF treatment). The mice injected with HVJ-E/MNPs were imaged using our magnetic particle imaging (MPI) scanner immediately (13 min) before, immediately (22 min) after, and 3, 7, and 14 days after the injection of agents, and the temporal changes of the average and maximum MPI pixel values in the tumor were quantitatively evaluated. The therapeutic effect was evaluated by calculating the relative tumor volume growth (RTVG) from the tumor volumes measured each day. Transmission electron microscopic (TEM) observation of resected tumors was also performed to confirm the intracellular distribution of MNPs. Results: The AMF treatment combined with BLM significantly decreased the RTVG value compared with AMF treatment alone at 9 to 14 days, and BLM alone at 3 to 5 days after AMF treatment. The average and maximum MPI pixel values in the tumor were almost constant for 14 days. TEM observation confirmed that most of the HVJ-E/MNPs were internalized into tumor cells within one hour after injection. Conclusion: A novel therapeutic strategy with use of AMF treatment and BLM was presented, and the time-dependent change of MNPs in tumors was evaluated using MPI. The present results suggest that this novel strategy can suppress tumor volume growth over AMF treatment or BLM alone, and can be performed repeatedly with a single injection of HVJ-E/MNPs. They also suggest that HVJ-E is effective for internalizing MNPs into cancer cells and that MPI allows for longitudinal monitoring of the distribution of MNPs in tumors.

新城疫病毒灭活苗免疫佐剂HVJ-E的研究

将SPF鸡分为4组,分别注射PBS、仙台病毒囊膜(HVJ-E)、新城疫病毒(NDV)灭活苗(KV)和HVJ-E+KV,于免疫后7、14和21d采血,测定血清中IFN-β、IFN-γ及抗NDV抗体的水平;免疫后21d以NDV强毒进行攻毒,攻毒后2、4、7和10d采集口咽腔、泄殖腔棉拭子分离病毒,以探讨HVJ-E作为NDV灭活苗佐剂的效果。结果表明:HVJ-E能显著提高NDV灭活苗的抗体产生水平并促进干扰素的分泌(P<0.05),添加HVJ-E佐剂的灭活苗的保护率为100%,排毒率为0%,显著提高免疫鸡的保护率。证明HVJ-E具有很好的免疫佐剂特性。

日本血凝病毒包膜的抗肿瘤应用策略研究进展

日本血凝病毒包膜(HVJ-E)可以通过增强肿瘤免疫反应、诱导肿瘤细胞凋亡以及靶向药物输送以达到高效治疗癌症的目的。诸多研究表明,HVJ-E的免疫治疗联合化疗(如与DTIC联用)、放疗(如与BNCT联用)、热疗(如将MNPs与HVJ-E结合)等能够展现出协同抗肿瘤作用。但是,由于HVJ-E全身给药易出现血凝问题,HVJ-E的应用受到限制。近年研究发现,通过LbL技术对其表面进行修饰,可以降低HVJ-E的血凝问题,并增加对肿瘤的特异性。该文将简述HVJ-E在肿瘤治疗上的应用策略,为促进HVJ-E在肿瘤治疗领域的进一步开发提供参考。