This study observed the effects of Guiqi Yiyuan Ointment(GQYY) on the left lung subjecting to bystander effect of right lung injury induced by ~(12)C~(6+) beam in rats and decipher the underlying mechanism from NOD-like receptor protein 3(NLRP3)/apoptosis-associated speck-like protein containing a CARD(ASC)/cysteinyl aspartate specific proteinase-1(caspase-1) pathway. Wistar rats were randomized into 7 groups: blank, model, inhibitor [200 mg·kg~(-1), N-acetylcysteine(NAC)], western drug [140 mg·kg~(-1) amifostine(AMI)], and high-, medium-, and low-dose(4.8, 2.4, and 1.2 g·kg~(-1), respectively) GQYY groups. The model of bystander effect damage was established by 4 Gy ~(12)C~(6+) beam irradiation of the right lung(with the other part shielded by a lead plate). The pathological changes in the lung tissue, the level of reactive oxygen species(ROS) in the lung tissue, and the levels of superoxide dismutase(SOD) and malondialdehyde(MDA) in the serum were observed and measured in each group. Furthermore, the mRNA and protein levels of NLRP3, ASC, caspase-1, and phosphorylated nuclear factor-κB p65(p-NF-κB p65)/nuclear factor-κB p65(NF-κB p65) were determined. Compared with the blank group, the model group showed thickened alveolar wall, narrowed alveolar cavity, and presence of massive red blood cells and inflammatory infiltration in the alveolar wall and alveolar cavity. In addition, the model group showed elevated ROS levels in both left and right lungs, elevated MDA level, lowered SOD level, and up-regulated mRNA and protein levels of NLRP3, ASC, caspase-1, and p-NF-κB p65/NF-κB p65. Compared with the model group, the drug administration in all the groups reduced inflammatory cell infiltration in the lung tissue. The inhibitor group and the western drug group showed enlarged alveolar cavity, thinned interstitium, and reduced inflammation. There was a small amount of alveolar wall rupture in the high-and medium-dose GQYY groups and reduced inflammatory cell infiltration in the low dose GQYY group. Compared with the model group, drug administration lowered level of ROS in the left and right lungs, lowered the MDA level, elevated the SOD level, and down-regulated the mRNA and protein levels of NLRP3, ASC, caspase-1, and p-NF-κB p65/NF-κB p65. GQYY can effectively reduce the damage caused by radiation and bystander effect, which may be associated with the ROS-mediated NLRP3 inflammasome activation.
Rats ; Animals ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; NF-kappa B/metabolism* ; Inflammasomes/metabolism* ; Lung Injury/genetics* ; Reactive Oxygen Species/metabolism* ; Bystander Effect ; Ointments ; Rats, Wistar ; Lung/metabolism* ; Caspase 1/metabolism* ; RNA, Messenger ; Superoxide Dismutase

OBJECTIVETo study the killing effects of the liposome-mediated thymidine kinase (TK)/ganciclovir (GCV) system on MG-63 osteosarcoma (OS) cells and its bystander effects.

METHODSLiposome-mediated TK gene transfected into MG-63 OS cells, the efficiency of transfection was analyzed by flow cytometry and observed under inverted fluorescence microscope. Non-transfected osteosarcoma MG-63 cells were divided into three groups,in the experimental group 1 transfected TK/GCV cells cultured in solutiona liquid mixture by supernatant by 1/10,1/7,1/5,1/2 ratio to original broth; in the experimental group 2 transfected cells cultured in solutiona liquid mixture of supernatant filtered through 0.22 microm filter by 1/10,1/7, 1/5, 1/2 ratio to original broth, in control group the transfection cells cultured in original culture solution. Cell growth inhibition rate and osteosarcoma cell sensitivity to TK/GCV system were measured by MTT assay in each group.

RESULTSThe TK gene was transfected into MG-63 OS cells successfully by liposome-mediated, flow cytometry instrument detection TK gene transfection cell transfection efficiency can reach 75.5%. Six days later the MTT assay showed that in the experimental group 1 inhibition rate of all concentration ratio of the mixed culture fluid were statistically significant as compared with the control group (P < 0.05), and in the experimental group 2 that of the 1/10 and 1/7 of concentration ratio of mixed culture medium was not statistically significant as compared with the control group (P > 0.05). TK gene transfected MG-63 cells increased with the the GCV concentration,the cell apoptosis rate increased.

CONCLUSIONThe experiment demonstrated that the MG-63 OS cells are sensitive to the liposome-mediated TK/GCV system and bystander effects are significant.

Apoptosis ; drug effects ; Bone Neoplasms ; enzymology ; genetics ; physiopathology ; Bystander Effect ; drug effects ; Cell Line, Tumor ; Cell Survival ; drug effects ; Ganciclovir ; toxicity ; Humans ; Osteosarcoma ; enzymology ; genetics ; physiopathology ; Thymidine Kinase ; genetics ; metabolism ; toxicity

Adipose-derived stem cells (ASCs) are believed to have potential use for treating many illnesses. Most cells, including ASCs, are generally cultured in medium containing fetal bovine serum (FBS). However, FBS, which could induce an immune response or infection, is not recommended for clinical applications. In the present study, we evaluated the morphology, proliferation rate, and characterization of rabbit ASCs grown in medium containing autologous serum (AS) and compared these cells to ones cultured with FBS. Morphological changes were monitored by microscopy and flow cytometry. Proliferation rates were assessed with cell counting and ASC phenotypes were characterized by flow cytometry using representative surface markers (CD44 and CD45). Expression of epidermal growth factor, brain-derived neurotrophic factor, and vascular endothelial growth factor was measured by reverse transcription-polymerase chain reaction. Results of our study showed that ASCs had a greater expansion rate in AS without developing morphological heterogeneity than cells grown in FBS. AS-cultured ASCs expressed representative growth factors, CD44 but not CD45, similar to cells cultured in FBS. Expression levels of some growth factors were different between AS and FBS. In conclusion, our findings indicated that AS could potentially be used as a culture medium supplement for the expansion of autologous ASCs.
Brain-Derived Neurotrophic Factor ; Bystander Effect ; Cell Count ; Epidermal Growth Factor ; Flow Cytometry ; Intercellular Signaling Peptides and Proteins ; Microscopy ; Phenotype ; Population Characteristics ; Stem Cells ; Vascular Endothelial Growth Factor A

OBJECTIVE: To investigate the mechanism and effect of diallyl disulfide (DADS) on the bystander effect of herpes simplex virus kinase/ganciclovir (HSV-tk/GCV) suicide gene therapy system which was mediated by recombinant adeno-associated virus (rAAV) in lens epithelial cells. METHODS: Immunohistochemistry method was used to determine the distribution of connexin 43 (Cx43) protein in cultured rabbit lens epithelial cells and rabbit lens epithelial cells transfected by HSV-tk suicide gene. Cx43 protein was measured and analyzed in cultured rabbit lens epithelial cells and rabbit lens epithelial cells transfected by HSV-tk suicide gene that was induced by various DADS. Cell survival was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. RESULTS: DADS increased the Cx43 protein expression in cultured rabbit lens epithelial cells and rabbit lens epithelial cells transfected by HSV-tk suicide gene, and especially in 50 μmol/L DADS. After combining with DADS, the bystander effect was significantly improved (P<0.05). CONCLUSION DADS can elevate the expression of Cx43 protein and enhance the bystander effect of HSV-tk/GCV suicide gene therapy system.
Adenoviridae ; genetics ; metabolism ; Allyl Compounds ; pharmacology ; Animals ; Antiviral Agents ; pharmacology ; Bystander Effect ; drug effects ; Cells, Cultured ; Connexin 43 ; metabolism ; Disulfides ; pharmacology ; Epithelial Cells ; metabolism ; Ganciclovir ; pharmacology ; Genes, Transgenic, Suicide ; Lens, Crystalline ; cytology ; drug effects ; metabolism ; Plant Oils ; Rabbits ; Simplexvirus ; enzymology ; Thymidine Kinase ; genetics ; metabolism

OBJECTIVETo investigate the potentiality of herpes simplex virus thymidine kinase transduced endothelial progenitor cells (EPC-TK) as angiogenesis-targeting vector in the glioma treatment in vitro and in vivo.

METHODSEPC-TK were mixed with human umbilical vein endothelial cells (HUVECs), U87 or U251 cells at various ratios for ganciclovir (GCV) treatment. The bystander effect was observed by counting the survival cells using MTT assay, and the apoptotic cells were determined by annexin-V and propidium iodide (PI) staining. EPC-TK, EPCs, or phosphate buffered saline (PBS) were injected into the nude mice model of glioma xenograft by tail vein, for the EPC-TK group, EPC group, and PBS group, respectively. And then the changes of tumor volume and tumor vasculature were observed.

RESULTSGCV killed most EPC-TK and reduced the number of other viable cells in a cell:cell ratio-dependent and time-dependent manner. EPC-TK obviously inhibited tumor growth. The tumor volumes on day 21 were 1741.20+/- 576.10 mm(3), 3275.52 +/- 710.86 mm(3) and 3033.09+/-1134.86 mm(3) in the EPC-TK, EPC and PBS group, respectively. EPC-TK also displayed a significant effect on the inhibition of tumor angiogenesis.

CONCLUSIONEPC-TK can exert a potent antiglioma effect via inhibiting angiogenesis.

Angiogenesis Inhibitors ; pharmacology ; Animals ; Antiviral Agents ; pharmacology ; Bystander Effect ; Cell Transformation, Viral ; physiology ; Endothelial Cells ; virology ; Endothelium ; Genetic Vectors ; Glioma ; therapy ; Humans ; Mice ; Mice, Nude ; Simplexvirus ; enzymology ; genetics ; Thymidine Kinase ; genetics ; Transduction, Genetic ; Transfection ; Xenograft Model Antitumor Assays

OBJECTIVETo investigate the enhancing effect of all-trans retinoic acid (ATRA) on the bystander effect of the herpes simplex virus thymidine kinase(HSV-TK)/ganciclovir (GCV) against androgen unresponsive prostate cancer.

METHODSThe bystander effect of the HSV-TK/GCV system was measured by methyl thiazolyl tetrazolium (MTT) assay on PC-3 cells before and after ATRA treatment. The growth and the histopathology of transplant tumors were observed in 4 groups of nude mice with prostate cancer.

RESULTSATRA augmented significantly the bystander effect of the HSV-TK/GCV system by reducing TK positive PC-3 cells from 50% to 30% (P < 0.05). HSV-TK showed an inhibiting effect, while ATRA with the HSV-TK/GCV system produced significant effect on prostate cancer 1 week earlier than the former (P < 0.05).

CONCLUSIONATRA can argument the in vivo and in vitro bystander effect of the HSV-TK/GCV system in the treatment of androgen unresponsive prostate cancer.

Animals ; Antineoplastic Agents ; pharmacology ; Bystander Effect ; drug effects ; Cell Line, Tumor ; Cell Survival ; drug effects ; Ganciclovir ; pharmacology ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; methods ; Humans ; Male ; Mice ; Mice, Nude ; Prostatic Neoplasms ; genetics ; pathology ; therapy ; Reverse Transcriptase Polymerase Chain Reaction ; Simplexvirus ; enzymology ; Thymidine Kinase ; genetics ; metabolism ; Tretinoin ; pharmacology ; Xenograft Model Antitumor Assays ; methods

OBJECTIVETo observe the targeted therapeutic effects of plasmid AF-pGL3-hTERT-TK on HepG2 cells.

METHODSHepG2 cells were cultured and pGL3-hTERT-TK and AF-liposome were constructed. HepG2 and L02 cells were transfected with AF-pGL3-hTERT-TK. The growth, apoptosis of the cells and the bystander effects were studied using liquid scintillation analysis and tunnel and flow cytometry.

RESULTSAfter the suicide gene was inserted into the downstream of hTERT, TK was effectively driven by the hTERT promoter, making the TK highly expressed in the HepG2 cells. The AF made the therapeutic gene enter the HepG2 cells more easily by recognizing and combining the ASGPR receptor protein on the HepG2 cell surfaces and induced their apoptosis and suicide with bystander effect. The apoptosis rate was 85%+/-3% in the HepG2 cells whereas in the normal L02 hepatic cells it was 16%+/-2%.

CONCLUSIONAF-pGL3-hTERT-TK can target and attack HepG2 cells and has almost no influence on normal L02 hepatic cells. AF-pGL3-hTERT-TK has a potential in the treatment of hepatocellular carcinomas.

Apoptosis ; Asialoglycoproteins ; Bystander Effect ; Fetuins ; Ganciclovir ; metabolism ; Genes, Transgenic, Suicide ; Genetic Therapy ; Hep G2 Cells ; Humans ; Telomerase ; metabolism ; Thymidine Kinase ; metabolism ; Transfection ; alpha-Fetoproteins

OBJECTIVETo study the antitumor and distant bystander effects of cationic liposome-mediated cytosine deaminase (CD)/5-fluorocytosine (5-FC) suicide gene system combined with interferon-gamma (IFN-gamma) in vivo.

METHODSMurine hepatoma 22 (H22) cells transfected by CD gene were inoculated subcutaneous in Kunming mice in the left axillary region, and the H22 cells without CD gene transfection were inoculated in the right axillary region. The mice were randomly divided into 4 groups and treated with normal saline , 5-FC, IFN-gamma, and 5-FC+ IFN-gamma on a daily basis. The tumor inhibition and distant bystander effects were observed in the mice.

RESULTSExposure of CD gene-transfected tumor to 5-Fc resulted in obvious tumor growth inhibition with an inhibition rate of 78.38%, which was significantly increased to 93.21% (P<0.01) with 5-Fc +IFN-gamma treatment. A notable distant bystander effect in the CD/5-FC suicide gene system was observed in vivo, with a tumor inhibition rate of was 54.42%; when combined with IFN-gamma, the inhibition rate increased significantly to 87.57% (P<0.05).

CONCLUSIONWhen combined with IFN-gamma, CD/5-FC suicide system has stronger anti-tumor and distant bystander effects. CD/5-FC suicide gene system combined with IFN-gamma may provide a potential therapy for malignant tumors.

Animals ; Bystander Effect ; Cations ; chemistry ; Cytosine Deaminase ; genetics ; Flucytosine ; pharmacology ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; methods ; Interferon-gamma ; therapeutic use ; Liposomes ; Liver Neoplasms, Experimental ; therapy ; Male ; Mice ; Random Allocation

OBJECTIVETo study the anti-glioma activity of treatment by bone marrow stromal cells (BMSCs) transfected with AdCMV-tk containing HSV-tk gene in rats.

METHODSPrimary cultured BMSCs were obtained and transfected with HSV-tk (BMSCs/tk) and were injected into contralateral brain of glioma-bearing rats to observe their tropism for glioma cells. RT-PCR was performed to examine the transduct of tk gene after it was transduced into BMSCs. C6 glioma cells were co-cultured with BMSCs transfected with HSV-tk. MTT test was performed to examine its antitumor activity. BMSCs, after being transfected with HSV-tk, were injected into contralateral brain tissue of glioma-bearing rats to show their in vivo antitumor activity. Dynamic MRI was performed to monitor the development of intracranial glioma.

RESULTSPurified BMSCs were obtained by primary cultured bone marrow cells. After being transfected with HSV-TK, the cells still stably displayed extensive tropism for intracranial glioma and transcripted tk gene. RT-PCR showed that BMSCs/tk were transduced tk gene obviously at 21 days after AdCMV-tk transfection. BMSCs/tk showed a clear bystander effect after being co-cultured with C6 glioma cells in vitro. TUNEL assay showed that BMSCs/tk could obviously show bystander effect and induce apoptosis of glioma cells in vivo with an apoptosis positive ratio of 20.38% +/- 2.57%, showing a statistically significant difference in comparison with BMSCs group (2.56% +/- 0.52%, P = 0.023) and control group (2.74% +/- 0.38%, P = 0.025). Compared with the control group (21.40 +/- 1.63 days), BMSCs/tk transplantation significantly prolonged the survival time of glioma-bearing rats (52.60 +/- 13.11 days, P = 0.000). MRI detection showed that the least volume of intracranial glioma in BMSCs/tk group (8.28 +/- 2.64 mm3), significantly smaller than that in BMSCs group (134.51 +/- 16.37 mm3, P = 0.001) and control group (147.22 +/- 31.05 mm3, P = 0.001). Some of the intracranial gliomaa disappeared after transplantation of BMSCs/tk.

CONCLUSIONBMSCs transfected with AdCMV-tk may become an effective therapy method in the treatment for glioma.

Animals ; Apoptosis ; Bone Marrow Cells ; cytology ; Brain ; pathology ; Bystander Effect ; Cell Line, Tumor ; Coculture Techniques ; Genetic Therapy ; methods ; Glioma ; pathology ; therapy ; Magnetic Resonance Imaging ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Simplexvirus ; enzymology ; Stromal Cells ; cytology ; enzymology ; transplantation ; Thymidine Kinase ; genetics ; metabolism ; Transfection

OBJECTIVETo investigate the effect of VEGF expression in osteosarcoma cell line and the target killing effect of HSV1-TK/GCV system on transfected osteosarcoma cells under hypoxia conditions.

METHODSEukaryotic expression plasmid with HRE promoter was constructed to express the antisense VEGF165 cDNA and Hygromycin phospho-transferase-thymidine kinase (HyTK) fusion gene. The recombinant vectors were then transfected into osteosarcoma cell line MG63 with lipofectin mediated gene transfer methods. PCR and RT-PCR were used to confirm the presence and expression of TK gene. The sensitivity of transfected cells to GCV and "bystander effect (BSE)" of HSV1-TK/GCV system under normoxia or hypoxia conditions were measured by MTT assay and mixed co-culture experiment. The expression of VEGF protein was detected by ELISA under hypoxia condition. Cell cycle phase distribution was determined by flow cytometry. In addition, electromicroscopy was used to document ultrastructural alterations.

RESULTSThe eukaryotic expression vector pBI-HRE-AsVEGF165 -HyTK was constructed successfully. The transfected cell line MG63TV was established and confirmed by PCR and RT-PCR of the presence of transgene and its mRNA expression. GCV was toxic to transfected cells in a concentration-dependent manner. The sensitivity to GCV toxicity was 100 times higher under hypoxia condition than that under normoxic condition. The mixed culture experiments showed that the "bystander effect" was enhanced significantly under hypoxia condition. VEGF expression of transgene cells under hypoxia condition decreased 50% compared to that of normal condition. Under hypoxia and GCV, DNA synthesis of MG63TV cells was inhibited along with an increase of cells at G0 approximately G1 phase, apoptosis and necrosis.

CONCLUSIONSAntisense VEGF expression driven by HRE promoter in combination with hypoxia can provide a target inhibition of VEGF expression in human osteosarcoma cells, with an enhanced selective killing effect and BSE of the HSV-TK/GCV system. The double-gene co-expression system in study provides experimental basis for therapy against osteosarcoma by a synchronous antiangiogenic and suicide gene approach.

Apoptosis ; Bone Neoplasms ; metabolism ; pathology ; Bystander Effect ; Cell Hypoxia ; Cell Line, Tumor ; Cell Proliferation ; DNA, Neoplasm ; biosynthesis ; Ganciclovir ; pharmacology ; Genetic Vectors ; Humans ; Hypoxia-Inducible Factor 1 ; genetics ; Oligodeoxyribonucleotides, Antisense ; Osteosarcoma ; metabolism ; pathology ; Phosphotransferases (Alcohol Group Acceptor) ; genetics ; Plasmids ; Promoter Regions, Genetic ; RNA, Messenger ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Thymidine Kinase ; biosynthesis ; genetics ; Transfection ; Vascular Endothelial Growth Factor A ; genetics ; metabolism