isolation-culture-adipogenic-induction-stromal-vascular-fraction

Isolation, Culture, and Adipogenic Induction of Stromal Vascular Fraction-derived Preadipocytes from Mouse Periaortic Adipose Tissue

Perivascular adipose tissue (PVAT) is an adipose tissue depot that surrounds blood vessels and exhibits the phenotypes of white, beige, and brown ...

Shanghai Jiao Tong University School of Medicine

The use of immunostimulatory molecule genes aiming at enhancing anti-tumor immunity has emerged as a new approach to treat cancers. 4-1BB signaling, an important costimulatory pathway delivering a signal for T cell activation, survival and growth, has become one of the most promising targets for cancer immunotherapy. In this work, a recombinant nonreplicative adenovirus (Ad.4-1BB scFv) carrying a single-chain Fv fragments (scFv) specific for 4-1BB gene (anti-4-1BB scFv) was generated, haracterized and explored for its stimulation of anti-tumor immunity in immunocompetent C57BL/6 mice. Ad.4-1BB scFv could efficiently infect murine hepatoma Hepa 1-6 cells and induce anti-4-1BB scFv expression on the cell surface. Moreover, Ad.4-1BB scFv did not cause obvious cytotoxicity effect on human and murine tumor cell lines (A549, PLC/PRF/5, Hepa 1-6 and TC-1) even at a high MOI, which suggested Ad.4-1BB scFv had no direct effect on tumor cells. Intratumoral injection of Ad.4-1BB scFv to established Hepa 1-6 tumors significantly suppressed the tumor growth in C57BL/6 mice. The anti-tumor effect might be mainly attributed to the anti-4-1BB scFv-mediated immune activity, as evidenced by enhanced interferon-gamma-producing splenic cells and increased lymphocytes infiltration in the tumor microenvironment. These results indicated that nonreplicative adenovirus carrying the anti-4-1BB scFv gene possessed powerful in vivo anti-tumor efficacy and might be a valuable tool for cancer immunotherapy.

Cancer gene therapy of adenovirus-mediated anti-4-1BB scFv in immunocompetent mice.

Virotherapy employing conditionally replicative adenovirus (CRAd) represents a novel targeted strategy for the hepatocellular carcinoma (HCC) treatment. In this study, we explored the potential influence of E3 region, which encodes several TRAIL-inhibiting proteins (E3-6.7K, E3-10.4K/14.5K and E3-14.7K), on CRAd mediated cytotoxicity to HCC cells. Two E1B-55 kDa-deleted CRAds containing E3 region (Ad.DeltaE1B) or no E3 region (Ad.DeltaE1B.DeltaE3) were fabricated. Ad.DeltaE1B.DeltaE3 exhibited higher cytocidal potency than Ad.DeltaE1B in all tested HCC cells (Hep3B, BEL-7404, BEL-7402, HuH7, PLC/PRF/5 and HepG2), suggesting that Ad.DeltaE1B.DeltaE3 mediated cytotoxicity was partly attributed to the absence of E3 region encoding TRAIL-inhibiting proteins. In representative Hep3B cells, Ad.DeltaE1B.DeltaE3 led to more drop of mitochondrial membrane potential (MMP) and much lower ATP level than Ad.DeltaE1B. Moreover, Ad.DeltaE1B.DeltaE3 induced early apoptotic cells and the late apoptotic/necrotic cells for three and four times more than those infected by Ad.DeltaE1B. The cytotoxicity to all TRAIL endogenously expressing HCC cells and MMP drop of Hep3B cells induced by Ad.DeltaE1B.DeltaE3 but not Ad.DeltaE1B could be significantly inhibited by z-vad-fmk, a pan caspase inhibitor, suggesting that the endogenous TRAIL-mediated apoptotic pathway may be implicated in the cytocidal potency of Ad.DeltaE1B.DeltaE3 on HCC cells although other unknown mechanisms may be also involved. Our findings provided the first evidence that CRAd without E3 region might be a smart choice for the virotherapy of HCC.

Influence of E3 region on conditionally replicative adenovirus mediated cytotoxicity in hepatocellular carcinoma cells.

Cancer gene therapy requires tumor-specific delivery and expression of a transgene to maximize antitumor efficacy and minimize side effects. In this study, we developed a new tumor-targeting, homologous recombination-based adenovirus vector system, HRAVS. HRAVS is composed of two adenovirus vectors, Ad.CMV.IR containing reverse sequence (IR) and a CMV promoter and Ad.IR.EGFP comprising the report gene EGFP and IR. For improved viral DNA replication and transgene expression, the E1a gene was added to HRAVS to generate the enhanced HRAVS, EHRAVS, which consists of Ad.CMV.IR and Ad.IR.EGFP/E1a. The optimal vector composition ratio of Ad.CMV.IR to Ad.IR.EGFP or Ad.IR.EGFP/E1a was identified as 30:70 based on EGFP expression efficiency in tumor cells. The transgene expression of HRAVS and EHRAVS was efficiently and specifically activated in tumor cells only and not in normal cells. Moreover, compared with HRAVS, EHRAVS infection led to higher virus yields and transgene expression and higher toxicity to tumor cells, and these results could be related to the involvement of E1a genes. The results in present study suggest the need for in vivo antitumor study using these new dual-Ad vector systems based on the homologous recombination.

Homologous recombination-based adenovirus vector system for tumor cell-specific gene delivery.

To develop a drug-eluting polymer film which can be easily personalized and rapidly made on the electrode array of a cochlear implant during surgery.

Development of a rapidly made, easily personalized drug-eluting polymer film on the electrode array of a cochlear implant during surgery.

To investigate the hearing protection outcomes of different drug-eluting analog electrode arrays implanted into guinea pig cochleae.

Hearing Protection Outcomes of Analog Electrode Arrays Coated with Different Drug-Eluting Polymer Films Implanted into Guinea Pig Cochleae.

To investigate the clinical characteristics and prognosis of nasopharyngeal carcinoma (NPC) in children and adolescents in different age groups.

Retrospective analysis of clinical features and prognosis of nasopharyngeal carcinoma in children and adolescents.

This study investigates the association between vestibular function and prognosis in patients with unilateral idiopathic sudden sensorineural hearing loss (UISSNHL).

Vestibular evoked myogenic potential may predict the hearing recovery in patients with unilateral idiopathic sudden sensorineural hearing loss.

Stroke prognosis is negatively associated with an elevation of serum bilirubin, but how bilirubin worsens outcomes remains mysterious. We report that post-, but not pre-, stroke bilirubin levels among inpatients scale with infarct volume. In mouse models, bilirubin increases neuronal excitability and ischemic infarct, whereas ischemic insults induce the release of endogenous bilirubin, all of which are attenuated by knockout of the TRPM2 channel or its antagonist A23. Independent of canonical TRPM2 intracellular agonists, bilirubin and its metabolic derivatives gate the channel opening, whereas A23 antagonizes it by binding to the same cavity. Knocking in a loss of binding point mutation for bilirubin, TRPM2-D1066A, effectively antagonizes ischemic neurotoxicity in mice. These findings suggest a vicious cycle of stroke injury in which initial ischemic insults trigger the release of endogenous bilirubin from injured cells, which potentially acts as a volume neurotransmitter to activate TRPM2 channels, aggravating Ca-dependent brain injury.

Article	Year
Cancer gene therapy of adenovirus-mediated anti-4-1BB scFv in immunocompetent mice. Cancer biology & therapy\| PubMed ID: 18614858	2008
Influence of E3 region on conditionally replicative adenovirus mediated cytotoxicity in hepatocellular carcinoma cells. Cancer biology & therapy\| PubMed ID: 19448394	2009
Homologous recombination-based adenovirus vector system for tumor cell-specific gene delivery. Cancer biology & therapy\| PubMed ID: 23792576	2013
Development of a rapidly made, easily personalized drug-eluting polymer film on the electrode array of a cochlear implant during surgery. Biochemical and biophysical research communications\| PubMed ID: 32220497	2020
Hearing Protection Outcomes of Analog Electrode Arrays Coated with Different Drug-Eluting Polymer Films Implanted into Guinea Pig Cochleae. Drug design, development and therapy\| PubMed ID: 34413631	2021
Retrospective analysis of clinical features and prognosis of nasopharyngeal carcinoma in children and adolescents. Frontiers in pediatrics\| PubMed ID: 36186630	2022
Vestibular evoked myogenic potential may predict the hearing recovery in patients with unilateral idiopathic sudden sensorineural hearing loss. Frontiers in neurology\| PubMed ID: 36408508	2022
Bilirubin gates the TRPM2 channel as a direct agonist to exacerbate ischemic brain damage. Neuron\| PubMed ID: 36921602	2023

Min Liang

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