离子通道门控动力学的随机建模

片膜钳技术为我们了解离子通道的特性奠定了实验基础,而根据片膜钳实验记录建立离子通道的门控数学模型是定量地刻画通道的门控行为和深刻理解其运行机制的必经途径。本文在简述用片膜钳技术记录离子通道电信号的科学背景之后,介绍了离子通道门控动力学随机建模的进展,并着重评论这一过程中出现的基本问题和富有特色的研究方法。

Effects of H pylori infection on gap-junctional intercellular communication and proliferation of gastric epithelial cells in vitro

AIM： To explore the effects of H pylori infection on gap-junctional intercellular communication （GJIC） and proliferation of gastric epithelial cells in vitro. METHODS： A human gastric epithelial cell line （SGC- 7901） cultured on coverslips was exposed overnight to intact H pylori （CagA^＋ or CagA^- strains） and sonicated extracts, respectively. GJIC between the cells was detected by fluorescence redistribution after photobleaching （FRAP） technique. Proliferation of SGC cells was determined by methylthiazolyl tetrazolium （MTT） assay. RESULTS： When compared with control in which cells were cultured with simple medium alone, both CagA^＋ and CagA^- H pylori isolates could inhibit GJIC （CagA^＋： F = 57.98, P 〈 0.01; CagA^-： F = 29.59, P 〈 0.01） and proliferation （CagA^＋： F = 42.65, P 〈 0.01; CagA^-： F = 58.14, P 〈 0.01） of SGC-7901 cells. Compared with CagA^- strains, CagA^＋ H pylori more significantly downregulated GJIC of gastric cells （intact Hpylori： t = 13.86, P 〈 0.01; sonicated extracts： t = 11.87, P 〈 0.01） and inhibited proliferation gastric cells to a lesser extent in vitro （intact H pylori： t = 3.06, P 〈 0.05; sonicated extracts： t = 3.94, P 〈 0.01）. CONCLUSION： Compared with CagA^- H pylori strains, CagA^＋ strains down-regulate GJIC of gastric epithelial cells more significantly and inhibit proliferation of gastric cells to a lesser extent in vitro. H pylori, especially CagA^＋ strains, may play an important role in gastric carcinogenesis.

Recombinant vascular basement-membrane-derived multifunctional peptide inhibits angiogenesis and growth of hepatocellular carcinoma

AIM： To investigate the anti-angiogenic and antitumor activities of recombinant vascular basement membrane-derived multifunctional peptide （rVBMDMP） in hepatocellular carcinoma （HCC）. METHODS： HepG2, Bel-7402, Hep-3B, HUVE-12 and L-02 cell lines were cultured in vitro and the inhibitory effect of rVBMDMP on proliferation of cells was detected by MTT assay. The in vivo antitumor efficacy of rVBMDMP on HCC was assessed by HepG2 xenografts in nude mice. Distribution of rVBMDMP, mechanism by which the growth of HepG2 xenografts is inhibited, and microvessel area were observed by proliferating cell nuclear antigen （PCNA） and CD31 immunohistochemistry. RESULTS： MTT assay showed that rVBMDMP markedly inhibited the proliferation of human HCC （HepG2, Bel-7402, Hep-3B） cells and human umbilical vein endothelial （HUVE-12） cells in a dose-dependent manner, with little effect on the growth of L-02 cells. When the ICs0 was 4.68, 7.65, 8.96, 11.65 and 64.82 μmol/L, respectively, the potency of rVBMDMP to HepG2 cells was similar to 5-fluorouracil （5-FU） with an IC50 of 4.59 μmol/L. The selective index of cytotoxicity to HepG2 cells of rVBMDMP was 13.8 （64.82/4.68）, which was higher than that of 5-FU [SI was 1.9 （8.94/4.59）]. The VEGF-targeted recombinant humanized monoclonal antibody bevacizumab （100 mg/L） did not affect the proliferation of HepG2, Bel-7402, Hep-3B and L-02 cells, but the growth inhibitory rate of bevacizumab （100 mg/L） to HUVE-12 cells was 87.6% ± 8.2%. AIternis diebus intraperitoneal injection of rVBMDMP suppressed the growth of HepG2 xenografts in a dose-dependent manner, rVBMDMP （1, 3, 10 mg/kg） decreased the tumor weight by 12.6%, 55.9% and 79.7%, respectively, compared with the vehicle control. Immunohistochemical staining of rVBMDMP showed that the positive area rates （2.2% ± 0.73%, 4.5%± 1.3% and 11.5% ±3.8%） in rVBMDMP treated group （1, 3, 10 mg/kg） were significantly higher than that （0.13% ± 0.04%） in the control group （P 〈 0.01）. The positive area rates （19.0% ± 5.7%, 12.2% ± 3.5% and 5.2% ±1.6% ） of PCNA in rVBMDMP treated group （1, 3, 10 mg/kg） were significantly lower than that （29.5% ± 9.4%） in the control group （P 〈 0.05）. rVBMDMP at doses of 1, 3 and 10 mg/kg significantly reduced the tumor microvessel area levels （0.26%± 0.07%, 0.12% ± 0.03% and 0.05% ± 0.01% vs 0.45% ± 0.15%） in HepG2 xenografts （P 〈 0.01）, as assessed by CD31 staining. CONCLUSION： rVBMDMP has effective and unique anti-tumor properties, and is a promising candidate for the development of anti-tumor drugs.

OPTIMAL CONDITIONS FOR DETECTING 5-BROMO-DEOXYURIDINE USED AS A MARKER OF TRANSPLANTED RETINAL PIGMENT EPITHELIAL CELLS

Objective To detemine optimal conditions by using 5 bromodeoxyuridine (BrdU) as a marker of transplanted retinal pigment epithelial (RPE) cells in the subretinal space of albino rabbits Methods Pigmented rabbit RPE cells at second to fifth passage were fed with 20 μmol/L BrdU in Eagle’s minimal essential medium (MEM) for five days After extensive wash with phosphate buffered saline (PBS),the cells were detached by trypsin and used for transplantation onto Bruch’s membrane of albino rabbits Eyes were enucleated at various times post transplantation Acetone, 4% paraformaldehyde, periodate lysine paraformaldehyde (PLP),or half strength Karnovsky’s fixatives were individually used to fix the tissue in order to find optimal condition for detecting BrdU marker The fixation was followed by embedding in OCT compound, glycol plastic, or paraffin The transplanted area was then sectioned, pepsin digested, and used for immunohistochemical staining with monoclonal antibody against BrdU and avidin biotin alkaline phosphatase complex (ABC AP) Results Frozen sections of acetone or paraformaldehyde fixed tissue gave strong immunostaining of BrdU but the overall morphology was poor Karnovsky’s fixed tissue offered strong staining but this was buried by strong background When using PLP as a fixative, we obtained strongly positive blue staining with very low background, and also excellent morphologic preservation Conclusion In combination with immunohistochemical detection method, BrdU labeling is an excellent long term marker for RPE transplantation one year after surgery To use BrdU as a marker necessitates the use of pepsin digestion to make the BrdU antigen in the nuclei accessible to antibody But the pepsin digestion may damage other tissues and result in overall poor morphology Among the fixatives tested in this study, PLP fixed tissues offered both strong BrdU staining and good preservation of structural integrity, particularly the fine structure of photoreceptor and RPE cells

In vivo detection of mucosal healing-involved histiocytes by confocal laser endomicroscopy

Histiocytes have a pivotal role in wound repair and intestinal epithelial recovery-the most important goal to sustain gut functionality.Yet,an in vivo description of colonic histiocytes by confocal laser endomicroscopy(CLE) is missing.Here,we report the case of a 45-yearsold male patient who was referred to our clinic with weight loss and a history of two consecutive Clostridium difficile colitis episodes,the latter cured 3 wk before present admission.Stool microbiology was negative.Conventional colonoscopy showed atrophy and a light mucosal oedema in the distal colon.During on-going endoscopy,we performed a fluorescein-aided CLE which revealed large polygonal(histiocytes-like) cells with copious cytoplasm and large nuclei in the lamina propria of the sigmoid colon as well as regenerative epithelial changes.Histopathological assessment of biopsies from the same areas confirmed the endomicroscopical findings:Periodic acid-Schiff-and CD68-positive foamy histiocytes in the colonic lamina propria and an advanced epithelial recovery.Since stool microbiology was repeatedly negative and polymerase chain reaction-analysis from colonic biopsies could not detect any mRNA for Thropheryma whippleii and common pathogens,we interpreted this particular setting as a mucosal healing process after consecutive Clostridium difficile infections.In conclusion,by describing these colonic histiocytes,we highlight the clinical usefulness of CLE in describing the entity of histiocytes in vivo and in real-time during the process of post-infectious mucosal healing in the colon.