Morphological changes of the peritoneum in peritoneal dialysis patients.

BACKGROUND: Long-term peritoneal dialysis (PD) requires that the peritoneal membrane remain effective for dialysis. Research directed toward human peritoneal morphology and structure is limited. The present study was performed to investigate morphological changes of the human peritoneal membrane during PD and to elucidate the possible mechanisms of its functional deterioration. METHODS: A total of 32 peritoneal biopsies were performed in normal subjects (n = 10), uremic nondialysis patients (n = 12) at the time of catheter insertion, and PD patients (n = 10) at the time of catheter removal or reinsertion or at the time of renal transplantation. Peritoneal morphology was examined by light microscopy, scanning electron microscopy, and transmission electron microscopy. RESULTS: The peritoneal membrane in normal subjects consisted of a monolayer of mesothelial cells on a basement membrane and a layer of connective tissue containing cells, blood vessels, and lymphatic vessels. Mesothelial cells were polygonal, often elongated, and had numerous microvilli on their luminal surface. There were lots of oval or roundish pinocytotic vesicles in the cytoplasm of the mesothelial cells. The peritoneal morphology of uremic nondialysis patients was similar to that of normal subjects. However, significant abnormalities of the peritoneal membrane were observed in PD patients, and the changes were found to be progressive. Microvilli were the first site of damage which involved microvilli shortening, a gradual reduction in their number, and, eventually, the total disappearance of microvilli. Mesothelial cells then detached from the basement membrane, disappearing completely in some cases. In the end, the peritoneal membrane consisted only of submesothelial connective tissue without any cells. CONCLUSIONS: PD can modify peritoneal morphology and structure. The morphological change is progressive and may be one of the important causes of peritoneal failure. Peritoneal biopsies can provide lots of valuable information about the effects of PD. Studying the relationship between peritoneal structure and its function proved very useful for understanding the physiopathology of the peritoneum during PD.

[Effects of hVEGF cDNA on random skin flap via a replication-deficient adenovirus vector].

OBJECTIVE: The purpose of this study was to determine the effects of local delivery of vascular endothelial growth factor( VEGF) transferred with adenovirus-mediated gene on the survival of ischemic random skin flap in rats. METHODS: The animals were divided into three groups randomly (n = 10) . A 2 cm x 8 cm dorsal skin flap was designed with the pedicle at the level of the iliac crest. In group A (AdCMV-VEGF), each animal received 10(12) PR replication-deficient recombinant adenovirus (AdCMV-VEGF) in the distal two-thirds of the proposed flap by means of the subdermal injection at ten different locations. In group B (AdCMV-GaI), each received 1012 PR AdCMV-Gal. In Group C (Saline), each received 1 ml saline. Three days after the treatment, the flap was elevated as planed way and re-sutured back to its donor site. All the animals were evaluated 7 days after the operation. RESULTS: The mean percentage of surviving flap area was (85.91 +/- 2.54)% in group A, (59.56 +/- l.18)% in group B, and (61.48 +/- l.09)% in group C. There was a significant increase in the percentage of the survival area in the flaps of the group A, compared with the group B and group C (Group B vs. Group A, P < 0.01; Group C vs.Group A, P < 0.01, Group B vs. Group C, P >0.05). Hybridization in the situ, the immunohistochemical stain showed that the VEGF was expressed in the survival tissue of the flap treated with the AdCMV-VEGF, but it was not found in the control groups. Histological analysis demonstrated qualitatively greater amount of granulation tissue and angiogenesis was found in the group treated with the AdCMV-VEGF than the controls. CONCLUSIONS: The results may indicate that Ad vector carrying VEGF cDNA could be useful in enhancing the survival of the skin flap due to the effect of the local delivery of the VEGF.

[Ionizing radiation-regulated killing of human hepatoma cells by liposome-mediated CDglyTK gene delivery].

EGR-1 gene promoter and CDglyTK gene were used to construct the pcDNA3-EGR-CDglyTK recombinant vector, in which CDglyTK gene expression was under the control of EGR-1 gene promoter. Cationic liposome LipofectAMINE was used to transfect plasmids into human hepatoma 7402 cell line. Subsequently, the transfected cells were treated with different doses of gamma-ray. Northern blot and Western blot showed that ionizing radiation can induce CDglyTK gene expression drived by EGR-1 promoter,in a dose-dependant manner. There was no ionizing radiation-inducible effect in pcDNA3-CMV-CDglyTK control group. Ionizing radiation also markedly enhanced the sensitivity of tumor cells transfected with pcDNA3-EGR-CDglyTK to prodrugs (GCV/5-FC) in tumor cell killing. The data indicated that EGR-1 promoter was inducible by ionizing radiation, whereas the CMV promoter was not. There was a synergetic effect between GCV and 5-FC. The cytotoxic effect of the suicide gene-ionizing radiation combination was stronger than suicide gene alone or ionizing radiation alone.

[Effect of soluble vascular endothelial growth factor (VEGF) receptor gene (sflt-1) and antisense VEGF nucleotide on neovascularization].

BACKGROUND & OBJECTIVES: Nutrients and growth factor provided by new blood vessel in tumor tissue is critical for fast growth of tumor, therefore, how to inhibit neovascularization and speedup neocrosis of tumor tissue is a promising way to treat tumor. This article emphasizes on both the blockage effect of soluble VEGF receptor gene (sflt-1) and antisense VEGF nucleotide on VEGF in tumor tissue, in order to inhibit its neovascularization. METHODS: Effect of antisense VEGF on VEGF secretion in MM45T. Li cells was observed after infection with Ad-antisense VEGF; Effect of recombinant soluble VEGF receptor protein (3' delta Flt-1) on proliferation of (human umbilical vascular endothelial cell) HUVEC induced by VEGF was investigated; Effect of both antisense VEGF and sflt-1 on neovascularization were tested after Ad-anti VEGF or Ad-sflt-1 injected in chorion of chicken embryo. RESULTS: VEGF concentration from infected MM45T. Li by recombinant Ad-antisense VEGF is only 15% of that from control group (P < 0.01); the proliferation of HUVEC induced by VEGF in condition medium containing 3' delta Flt-1 is significantly reduced, and negatively related with dose within certain range; Both sflt-1 and antisense VEGF nucleotide obviously inhibited formation of new blood vessel, even caused dead embryo. CONCLUSION: Both sflt-1 and antisense VEGF could effectively inhibited neovascularization separately, combination therapy can enhance the inhibitory effect, but the mechanism is different.

[Screening and identification of tumor related genes in early stages of transformed NIH3T3 cells associated with overexpressed PTPalpha].

An inducible-PTPalpha expression system was used to screen differential expression genes in NIH3T3 cells overexpressing PTPalpha, which could be used as a model for studying mechanism of tumorigenesis at early stage. After PTPalpha induction for 24 h, 65 differential bands were obtained, among which, there were 29 known genes, 12 known EST, 6 novel EST by bioinformatic analysis. Functions of the known genes were related to signal transduction, structural/adhesion, energy metabolism, transcription/translation, ribosome and apoptosis. The differential expression of the genes coding for G protein, TCR, Trx-related protein, caveolin-1 was verified by RT-PCR and Northern blot. The results showed that the expression of numerous genes was changed after the induction of PTPalpha for 24 h, these genes are related to many cellular physiological functions, and some of them play important roles in the early stage of tumorigenesis. The results provide basis for further exploring the molecular mechanism of tumorigenesis and these genes may well be candidate targets for gene therapy.

Screening and identification of gastric adenocarcinoma metastasis-related genes by using cDNA microarray coupled to FDD-PCR.

To clone gastric adenocarcinoma metastasis related genes, RF-1 cell line (primary tumor of a gastric adenocarcinoma patient ) and RF-48 cell line (its metastatic counterpart) were used as a model for studying the molecular mechanism of tumor metastasis. Two fluorescent cDNA probes, labeled with Cy3 and Cy5 dyes, were prepared from RF-1 and RF-48 mRNA samples by reverse transcription method. The two color probes were then mixed and hybridized to the cDNA chip constructed by double-dots of 4 096 human genes, and scanned at two wavelengths. The experiment was repeated for 2 times. Differential expression genes from the above two cells were analyzed using the computer. 138 in all genes (3.4%) revealed differential expression in RF-48 cells compared with RF-1 cells: 81(2.1%) genes revealed apparent up-regulation, and 56(1.3%) genes revealed down-regulation. 45 genes involved in gastric adenocarcinoma metastasis were cloned using fluorescent differential display-PCR (FDD-PCR), including 3 novel genes. There were 7 differential expression genes that agreed with each other in two detection methods. The possible roles of some differential expressed genes, which maybe involved in the mechanism of tumor metastasis, were discussed. cDNA chip was used to analyze gene expression in a high-throughput and large scale manner, in combination with FDD-PCR for cloning unknown novel genes. In conclusion, some genes related to metastasis were preliminarily scanned, which would contribute to disclose the molecular mechanism of gastric adenocarcinoma metastasis.

[Changes of phenotypes associated with activated Src by overexpressed PTPalpha in NIH3T3 cells].

NIH3T3 cells transfected with inducible expression vectors harboring the protein tyrosine phosphatase alpha (PTPalpha) gene were used as a model to study the mechanism of tumor formation, by which the changes of phenotypes in inducible cells were studied. When NIH3T3 cells transfected with PTPalpha were induced for 24 hours, the expression levels of PTPalpha in inducible cells was higher than those in uninducible cells as judged by RT-PCR and Western blotting; the expression level of Src in inducible cells was the same as those in uninducible cells, but activity of Src kinase in inducible cells was higher than those in uninducible cells.The level of phosphated tyrosine in Src was reduced in inducible cells. The malignant changes of phenotypes in the inducible cells were verified by flow cytometry and electron microscope. The results show that 24 h induction of PTPalpha could initiate the transformation of NIH3T3 cells transfected with PTPalpha inducible expression vectors, and the transformation may be associated with activated Src where pTyr(527) in the C tail was dephosphorylated by increased expression level of PTPalpha in the cells.

[Combination therapy of experimental murine hepatoma with mIL-12 gene and MHC I gene mediated by liposome].

BACKGROUND & OBJECTIVE: Interleukin-12 gene and MHC I gene have been used in gene therapy for cancer individually. To explore the synergistic antitumor effects of these two genes, the therapeutic effects of mIL-12 gene combined with MHC I (mouse H-2K) gene mediated by cationic liposome for experimental murine hepatoma were investigated. METHODS: Balb/C mouse liver cancer MM45T. Li cells were transfected with pcDNA3/mIL-12, pcDNA3/H-2Kb(H-2K of C57BL/6 mouse), and pcDNA3/GFP (reporter gene) mediated by lipofectAMINE 2000(LF 2000). The expressions of foreign genes in transfected cells were detected. Balb/C mice were inoculated subcutaneously with pcDNA3/mIL-12 and pcDNA3/H-2Kb transfected MM45T. Li. The tumorigenesis of the inoculated cells was detected. After intratumoral injection with LF2000-plasmid DNA complexes, the growth of murine tumor and the survival time of the tumor bearing mice were observated. RESULTS: The optimal ratio of LF2000: DNA is 3:1(microgram: microgram). The transfection efficiency reached to 30%. RT-PCR result showed the specific amplified fragments of the mIL-12 cDNA and H-2Kb cDNA in the transfected cells. Western blot analysis showed the expression of H-2Kb protein at 57 kDa. ELISA assay showed that the secretory mIL-12 was 48 ng/ml/10(6) cells. The tumorigenesis was decreased for transfected MM45T. Li cells with pcDNA3/mIL-12 and pcDNA3/H-2Kb. FACS assay showed that the numbers of CD3+, CD4+, and CD8+ cells from murine spleen were increased more in therapeutic group than in control group. The tumors grow slowly. The mIL-12 gene combined with H-2Kb gene has stronger antitumor effect for mouse liver cancer than single gene. CONCLUSION: The combination therapy with mIL-12 gene and MHC I gene mediated by LF-2000 have the positive synergistic antitumor effect for experimental murine hepatoma.

The Expression of Adenoviral Mediated gfp Reporter Gene in Tumor Cells Induced and Regulated by Irradiation.

In order to establish a radiation inducible gene expression system for cancer gene therapy, the promoter sequence of radiation-inducible Egr-1 gene was amplified from genomic DNA of BALB/c mouse with PCR method, and linked to gfp reporter gene. Then the pEgr-gfp expression cassette was subcloned into an adenoviral shuttle plasmid to generate recombinant adenovirus of AdEgr-GFP by using a novel, high efficient method of homologous recombination in bacteria. After infection with AdEgr-GFP, MM45T.Li tumor cells were exposed to different doses of gamma-irradiation from 0 Gy to 15 Gy in vitro. The percentage of GFP expression positive cells increased greatly in a dose-dependent manner as detected by FACS and Western blot analysis. For in vivo study, AdEgr-GFP were injected intratumorally, and tumor site received different doses of local gamma-irradiation 48 h after injection, and after 8 h the tumor samples were biopsed for investigating the GFP expression. Tumor tissue image analysis revealed that gamma-irradiation could markedly increase GFP expression in a dose-dependent manner as compared with that of non-irradiated control group. Our results indicate that the irradiation can effectively control adenoviral-mediated GFP expression in tumor cells via Egr-1 promoter, and these data laid basis for further gene radiotherapy study.

Construction of a Helper-dependent Adenoviral Vector and Its application to Mice.

First-generation adenoviral vector with E1 and E3 deleted can effectively deliver foreign genes into target cells and leads to high level of expression of transgenes, but it leads only to transient expression because of the cellular immunity against early and late viral antigens expressed in targeted cells. In order to overcome these difficulties, we have recently developed a helper-dependent recombinant adenovirus vector HAdI-hFVIII in which a large part of the viral genome, including l3, L1, L2, VAI-VAII and pTP regions, was deleted. The novel viral vector DNA can be effectively packaged, amplified in 293 packaging cells co-transfected with an E1-substituted AdI-hFVIII in trans and can be easily separated from helper virus by gradient centrifugation. The hFVIII expression was found in mice after intravenous injection of the new vector. The duration of hFVIII protein in plasma of mice was prolonged compared to that of AdI-hFVIII after injection. It suggests that the novel vector could be less immunogenic in vivo.

The Construction and Expression of Polycistronic Retroviral Vector Carrying Genes of TNF-alpha, IL-2 and NeoR.

The coordinate expression of a marker gene and therapeutic genes in a single vector is markedly advantageous. The internal ribosome entry site (IRES) from encephalomyocarditis and that from polio viruses were used to construct a polycistronic retroviral vector pGCEN/TRI, where Neo-resistant gene was used as a marker gene and TNF-alpha cDNA, IL-2 cDNA as genes of interest, so that the LTR promoter derived the expression of a tricistronic mRNA. Amphotropic packaging cells PA317 transfected with pGCEN/TRI using LipofectAMINE was selected with G418 and the positive clones expressing the genes of interest to produce high-titer retrovirus (5x10(5) CFU/ml) were obtained. PCR confirmed the presence of proviral DNA in the positive producer cells and Northern blot analysis revealed a single, LTR promoted transcript. The transduced cells expressed TNF-alpha and IL-2 at different levels. This demonstrated that polycistronic retroviral vector containing IRES could efficiently express multiple therapeutic genes in the same target cell.

Transduction of human hepatocellular carcinoma cells with human alpha-interferon gene via retroviral vector.

AIM:To investigate the therapeutic potential of gamma interferon (IFN-alpha) genemodified human hepatocellular carcinoma (HCC) cells.METHODS:The IFN-alpha gene was introduced retrovirally into four HCC cell lines.Secreted IFN-alpha activity was assessed using bioassay. The expression of MHC molecules was detected by FACS.Tumorigenicity was analysed by tumor formation in nude mice.RESULTS:Four IFN-alpha gene transduced HCC cell lines secreted different amounts of IFN-alpha, as in the same case of five clones derived from one HCC cell line. Transduction with IFN-alpha caused significant increase in the expression of major histocompatibility complex (MHC) antigens on HCC cells. The expression of HLA class I was increased by 2-3 times in terms of mean fluorescence intensities, while for class II expression, the percentage of positive cells augmented from < 10% to &lg 50%. When equal amount of tumor cells were injected into nude mice, the tumor igenicity some transduced cells decreased dramantically.CONCLUSION:IFN-alpha gene transduction can convert weakly imunogenic HCC cells to activate antitumor immune response, and further pave the way for the future use of such gene modified tumor cells as a modality for the cancer immunotherapy.