Background Channelrhodopsin-2 (ChR2)is a cation channel isolated from the eyespot of Chlamydomonas algae and has been used to control neuron activity.The light stimulation is a more precise fashion whether space or time than that of electrical,magnetic and ultrasound stimulation. Objective This study was to construct a replication deficient recombinant adenovirus cxpression vector of ChR2 and to determine its function.Methods Human embryo kidney 293 (HEK293) cell line was cultured and passaged in DF12 medium containing 10％ fetal bovine serum(FBS).The ChR2 gene was cloned at the downstream of cytomegalovirus(CMV)promoter of the adenoviral shuttle plasmid pSB291 in sense direction,and the resultant recombinant plasmid pSB291-hChR2- GFP was transfected into HEK293 cell together with plasmid pBHG lox ( deltaE1,3 ) containing adenoviral genome,then small amounts replication deficient recombinant adenovirus expression vector of ChR2 (Ad-ChR2) was obtained.Through amplification gradient centrifugation and dialysis,pure Ad-ChR2 was obtained.Visual cortex cells derived from 4 1-day-old clean Long Evans rats were primary cultured with serum-free culture media and infected by AdChR2.When expressing green fluorescencc,those cells received the stimulated of blue light with 460 nm.Patch clamp technique was applied to record an action potential. Results After purification and concentration,the titer of AdhCHR2 reached 7.9×1010 PFU/ml.Twenty-four hours after transfect of Ad-ChR2,HEK293 cell membrane showed the green fluorescence for the recombinant plasmid with green fluorescence protein under the inversed fluorescence microscope.The HEK293 cells change their shape from flat to round 13 days after transfected.The primary cultured visual cortex cells exhibited the green fluorescence 3-5 days after infected by Ad-ChR2.The action potentials evoked by blue light stimulation were recorded with patch clamp on those cells expressing green fluorescence. Conclusions Ad-ChR2 expressing vector is constructed successfully in this study.It is verified that Ad-ChR2 expressing vector can infect visual cortex cells with visual function.This result is very important for visual plasticity study.

OBJECTIVETo investigate the expression of guanylin in colorectal cancer.

METHODSThe expression of guanylin was examined by RT-PCR and semiquantitative analysis in 20 cases of colorectal cancer, and its relationship with clinical characteristics was analyzed.

RESULTSThe positive expression of guanylin in normal tissue (80%, 16/20) was significantly higher than that in tumor tissue (35%, 7/20) (P<0.01). The same result was found in the semiquantitative analysis of 14 cases with differential expression. Differential expression of guanylin in colorectal cancer was associate with TNM stage (P<0.05), not with sex, Borrmann type and degree of differentiation (all P>0.05).

CONCLUSIONThere is differential expression of guanylin in colorectal cancer, and this kind of differential expression is associated with tumor TNM stage.

Colorectal Neoplasms ; metabolism ; pathology ; Female ; Gastrointestinal Hormones ; metabolism ; Humans ; Male ; Middle Aged ; Natriuretic Peptides ; metabolism ; Neoplasm Staging

OBJECTIVETo investigate the anti-tumor effect of adenovirus-mediated Bcl-XL shRNA on colon cancer cells in vitro.

METHODSA recombinant Bcl-xl adenovirus was constructed, amplified, and purified. The effect on mRNA expression of Bcl-XL was assessed by RT-PCR, and the effect on apoptosis-induction of colon cancer(Lovo cell line) in vitro was assessed by MTT assay and cell clonogenic assay.

RESULTSRT-PCR showed that Ad/Bcl-XL shRNA significantly down-regulated the mRNA expression of Bcl-XL in Lovo cells. Ad/Bcl-XL shRNA suppressed the proliferation of Lovo cells in a dose-dependent as well as a time-dependent manner compared with Ad/GFP (P<0.05). Treatment with Ad/Bcl-XL shRNA dramatically suppressed the colony formation of Lovo cells in a dose-dependent manner (P<0.05). Ad/Bcl-XL shRNA showed no effect on normal human fibroblast.

CONCLUSIONAd/Bcl-XL shRNA exhibits cytotoxic effect on Lovo cells and may have the potential value in the treatment of colon cancer.

Adenoviridae ; genetics ; Cell Line, Tumor ; Cell Proliferation ; Colonic Neoplasms ; metabolism ; pathology ; Humans ; RNA, Small Interfering ; genetics ; bcl-X Protein ; genetics ; metabolism

OBJECTIVETo compare the effect of Acanthopanacis senticosi injection, theophylline and caffeine on human sperm mobility in vitro.

METHODSWe incubated the sperm aseptically obtained by masturbation from 12 asthenospermia men and treated by swim-up technique in Acanthopanacis senticosi injection (10 g/L), theophylline (3 mmol/L) and caffeine (7 mmol/L) respectively, and detected various sperm parameters with the computer-assisted sperm analysis (CASA) system at 0 h, 1 h and 3 h.

RESULTSAcanthopanacis senticosi injection significantly increased sperm motility, the percentage of progressive motile sperm, straight line velocity (VSL) and curvilinear velocity (VCL) as compared with theophylline and caffeine (P < 0.05).

CONCLUSIONAcanthopanacis senticosi injection can activate the mobility of human sperm in vitro.

Caffeine ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Eleutherococcus ; chemistry ; Humans ; In Vitro Techniques ; Male ; Sperm Motility ; drug effects ; Theophylline ; pharmacology

OBJECTIVETo investigate the chromosomal aberration in sporadic colorectal carcinoma and its association with clinicopathological features.

METHODSComparative genomic hybridization(CGH) was used to screen the changes in the number of DNA sequence copies in 40 sporadic colorectal cancer patients in order to identify regions that contain genes important for the development and progression of colorectal cancer.

RESULTSIn 40 sporadic colorectal cancer, frequent gain at 20 q, 12 q, 13 q, 7 p, 7 q and 16 q were found, while loss was also found at 18 q, 5 q, 4 q, 8 pand 17 p. The number of chromosomal aberration was closely associated with tumor stage(P<0.05). No significant association was found between the number of chromosomal aberration and tumor site, histopathologic type and histologic grade.

CONCLUSIONSChromosomal aberration exists generally in sporadic colorectal carcinoma. The number of chromosomal aberration and gain of 20q are closely associated with tumor stage.

Adult ; Aged ; Aged, 80 and over ; Chromosome Aberrations ; Chromosome Mapping ; Colorectal Neoplasms ; genetics ; pathology ; Comparative Genomic Hybridization ; DNA Probes ; Female ; Gene Dosage ; Humans ; Male ; Middle Aged ; Neoplasm Staging ; Oligonucleotide Array Sequence Analysis

Objective To investigate the relationship between apolipoprotein E (APOE) genotype and onset of hypertensive intracerebral hemorrhage (HICH). Methods One hundred and twenty-eight patients with HICH,admitted to our hospital from September 2007 to December 2010,and 84 healthy controls clinically flee from neurology disease were chosen in our study.A POE genotype was determined using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP); the distribution ofAPOE genotype and allele fiequency between the 2 groups were compared; the age of onset,gender,blood pressure,blood fat,bleeding part and size of haematoma in patients with different APOE genotype were further studied. Results The subjects with smoking hobby,diabetes history and higher concentrations of TC and LDL-C in the HICH group were obviously more than those in the control group (P＜0.05).As compared with those in the controls,the distributions of the APOEε3/4 and APOEε4 in the HICH group were significantly higher, and the distributions of the APOEε3/3 and APOEε3 in the HICH group were significantly lower (P＜0.05). The APOEε3/4 and APOEε4 carriers enjoyed higher percentages of onset age less than 45 years and superficial haematoma than A POEε3/3 and the A POEε3 carriers. The onset systolic blood pressure in APOEε3/4 and APOEε4 carriers was obviously lower than that in A POEε3/3 and A POEε3 carriers (P＜0.05). The plasma concentrations of LDL-C and TC in APOEε3/4 and APOEε4 carriers was significantly higher than those in APOEε3/3 and APOEε3 carriers (P＜0.05). Conclusion The APOE genotype influences the onset of HICH, and its influence factors included onset age, blood pressure,blood fat and bleeding part,which may concern with the cerebral artherosclerosis or amyloidosis.

Objective To evaluate the clinical effects of the bone-ring augmentation in implant restoration using the bone ring technique.Methods Sixteen patients (30 teeth) with severe alveolar bone absorption were selected.Bone rings (circular osteotomies) were outlined at the symphysis area and a central osteotomy for implant placement was done using the bone ring drills before its removal.The rings were then removed and sculptured to fit the alveolar ridge and then covered by Bio-oss and Bio-Gide membrane.This was followed by screwing the implant through the ring,gaining its primary stability from the prepared basal bone.After 4-6 months,the implant was exposed and abutment connected for temporary restoration.Results Of the 16 patients,14 cases were healed by first intention,2 cases were infected after the bone ring implantation resulting in some bone absorption but healed after bone grafting materials were placed at the second stage of the onlay procedure.The bone ring implantation showed no absorption and no implant failure.After the bone grafting the alveolar bone height increased by 4.1 mm on average and the thickness increased by 3.27 mm on average.Conclusions Implant bone ring can effectively augment the absorbed alveolar ridge.The procedure has the advantages of bone removal,grafting and implantation at one stage.

OBJECTIVETo investigate the clinical characteristics, diagnosis, treatment and prognosis of deep sarcoma of the penis.

METHODSThe pathological and clinical data of 2 cases of deep sarcoma of the penis were analyzed retrospectively and the literature reviewed.

RESULTSBoth of the cases were treated by total penectomy. Epithelioid angiosarcoma of the penis was confirmed by postoperative pathology in one patient, who died of pulmonary metastasis in the eighth month after the operation; and epithelioid sarcoma of the penis was confirmed in the other, who died of brain metastasis in the second month after the operation.

CONCLUSIONDeep sarcoma of the penis is rare but can be diagnosed pathologically. Total penectomy is the main option for its treatment. Node dissection, with poor prognosis, is not recommended unless adenopathy is palpable.

Fatal Outcome ; Humans ; Male ; Middle Aged ; Penile Neoplasms ; diagnosis ; surgery ; Prognosis ; Retrospective Studies ; Sarcoma ; diagnosis ; surgery

OBJECTIVETo investigate the therapeutic efficacy of double-mutated oncolytic adenovirus AxdAdB-3 in combination with gemcitabine for treating bladder cancer in an orthotopic nude mouse model.

METHODSThe susceptibility to the adenovirus was evaluated in bladder cancer cell lines YTS-1, T24, 5637 and KK47, and normal cell lines HCV29 and WI38. The cells were infected with AxCAlacZ and stained with 5-bromo-4-chloro-3-indolyl-β-galactoside (X-Gal). Immunostaining against adenoviral hexon protein was performed to determine the selective replication of AxdAdB-3 in the cancer cells. Flow cytometry was used to determine the YTS-1 cells in S phase of cell cycle after adenovirus infection. Cell viability after AxdAdB-3 and/or gemcitabine was measured by CCK-8 assay. Orthotopic bladder cancer model was established in nude mice, and the inhibitory efficacy of intravesical instillation therapy with AxdAdB-3 or/and gemcitabine was assessed.

RESULTSGene transduction efficiency was different among the cell lines, and correlated with expression of CAR. 5637 and KK47 cells with high expression of CAR were more susceptible to the adenovirus, whereas YTS-1 and T24 cells with little CAR expression were resistant to adenoviral infection. Immunostaining showed that the expression levels of hexon protein varied among the cell lines. Normal cells infected with AxdAdB-3 expressed little hexon protein. The proportion of S-phase cells was (39 ± 3) % and (49 ± 5) % in the AxCAlacZ- and AxdAdB-3-infected bladder cancer cells, respectively. AxdAdB-3 effectively induced S-phase entry of cell cycle (P < 0.05). AxdAdB-3 combined with gemcitabine significantly inhibited the growth of bladder cancer cell lines. In vivo, the mean weight of the bladder tumors in mice treated with intravesical instillation of AxCAlacZ, gemcitabine, AxdAdB-3, and AxdAdB-3 + gemcitabine were 400.6, 126.4, 82. 0, 40.4 mg, respectively. Either AxdAdB-3 (P < 0.0001) and gemcitabine (P < 0.0001) suppressed the tumor growth in nude mice, and the combination therapy reduced tumors more effectively than either AxdAdB-3 (P < 0.0001) or gemcitabine (P < 0.0001) alone.

CONCLUSIONSIntravesical instillation therapy with AxdAdB-3 in combination with gemcitabine can effectively inhibit the orthotopic bladder cancer in nude mouse, and further relevant clinical studies are guaranteed.

Adenoviridae ; genetics ; Administration, Intravesical ; Animals ; Antimetabolites, Antineoplastic ; administration & dosage ; pharmacology ; therapeutic use ; Antineoplastic Combined Chemotherapy Protocols ; administration & dosage ; pharmacology ; therapeutic use ; Deoxycytidine ; administration & dosage ; analogs & derivatives ; pharmacology ; therapeutic use ; Galactosides ; Indoles ; Mice ; Mice, Nude ; Models, Animal ; Urinary Bladder Neoplasms ; drug therapy

OBJECTIVETo investigate the effect of electroporation-mediated gene transfect on the expression of cyclins during mandible distraction in rabbit.

METHODSBilateral mandibular osteotomy was performed in 45 New-Zeland rabbits. After a latency of 3 days, the mandibles were elongated using distractors with a rate of 0.8 mm/day for 7 days. After the completion of distraction, the rabbits were randomly divided into 5 groups. 2 microg (0.1 microg/microl) of pIRES-hVEGF165-hBMP2, recombinant plasmid pIRES-hBMP2, recombinant plasmid pIRES-hVEGF165, pIRES and the same volume of normal saline (NS) was injected into the distraction area in each group, respectively. After injection, electroporation was performed in every group. Three animals in each group were sacrificed at 7, 14, and 28 days after completion of distraction, respectively. The lengthened mandibles were harvested and processed for immunohistochemical examinations. The expression of cyclins A, D1 ,E in positive cells were measured by CMIAS-2001A computerized image analyzer. The data were analyzed with the single factor analysis of variance and q test.

RESULTSCyclins A, D1, E staining was mainly located in inflammatory cells, granulation tissue monocyte, fibroblast, osteoblasts, osteocyte and the connective tissues around the new bone. The expression reached to the peak at 7th day of consolidation, and decreased at 14th day, and weak at 28th day. Image analysis results showed that, at 7th day, the expression absorbance A in group C (0.59 +/- 0.14) was the strongest, compared to group A (0.41 +/- 0.13), B (0.38 +/- 0.14), D (0.34 +/- 0.12) and E (0.31 +/- 0.10), showing a significant difference (P < 0.05, P < 0.01). There was no significance difference between group A and B (P > 0.05), but the difference between group A/B and group D/E (P < 0.05). At 14th and 28th day, there was no significant difference among group A (0.39 +/- 0.11), B (0.34 +/- 0.10) and C (0.33 +/- 0.09) (P > 0.05), but there was significant difference between group A/B/C and group D (0.19 +/- 0.12) or E (0.14 +/- 0.04) (P < 0.05 or P < 0.01).

CONCLUSIONSElectroporation-mediated gene transfection can promote cyclins A, D1, E expression effectively, which may promote cell differentiation and proliferation, stimulate extracellular matrix synthesis and new bone formation in distraction gap.

Animals ; Cyclins ; metabolism ; Electroporation ; Genetic Therapy ; Mandible ; surgery ; Osteogenesis, Distraction ; methods ; Plasmids ; Rabbits ; Transfection