Objective:To design and construct the replication-deficient recombinant adenovirus Ad-siCTGF which can silence the expression of connective tissue growth factor (CTGF) by RNA interference and verified its function. Methods:A specific sequence, which was verified to be able to silence CTGF gene with high efficiency, was cloned into pSES-HUS vector to produce the shuttle plasmid pSES-siCTGF. The plasmid after Pme Ⅰ linearization was cotransduced with pAdEasy into BJ5183 E.coli strains to construct recombinant vector Ad-siCTGF. After linearization treatment with Pac Ⅰ enzyme digestion Ad-siCTGF was transfected into HEK293 cells via liposome mediation. The recombinant adenovirus was packaged. The titer of the Ad-siCTGF was increased after three times of cross-infection. 4T1 cells were infected with the adenovirus. The silencing efficiency was tested by real-time fluorescence quantitative (RFQ)-PCR and Western blotting.Results:Pac Ⅰ enzyme digestion electrophoresis indentified that recombinant adenovirus was successfully constructed. The titer of the recombinant adenovirus Ad-siCTGF was 2.6×10~(10) pfu/mL after amplification and purification. The CTGF mRNA and protein expression in 4T1 cells were decreased by 36.27% and 31.56%, respectively, compared with the control groups.Conclusion:The recombinant adenovirus which can silence the expression of CTGF was successfully constructed. It laid a good foundation for further investigation of the action mechanism of CTGF in tumor cells.

OBJECTIVEUsing rats fed in intermittent hypoxia environment to study the relationship between sleep apnea hypopnea syndrome (SAHS) of children and growth retardation.

METHODSThe hypoxic chamber was designed and manufactured, the control of intermittent hypoxia was achieved. Twenty-four rats were randomly divided into three groups: mild and severe hypoxia group, and control group. In control group, the animals were normally fed, without interruption. The animals in other two groups were kept in the cabin, simulated mild and severe intermittent hypoxia conditions 8-hour a day, a total of 35 days. According to the results of preliminary experiments, the concentration of intermittent hypoxia and frequency were determined. The animals with mild hypoxia events occurred nearly six times per hour, the average minimum oxygen saturation dropped to 0.853, the animals with severe hypoxia events occurred nearly 24 times per hour, the average minimum oxygen saturation dropped to 0.776. Body mass and length were measured before and after experiment. The serum insulin-like growth factor (IGF)-1 and insulin-like growth factor binding protein (IGFBP)-3 expression were tested from venous blood by enzyme-linked immunosorbent assay (ELISA).

RESULTSThe length and body mass of rats in three groups before and after experiment were not statistically different (P>0.05). Before the experiment the serum IGF-1 and IGFBP-3 levels were not significantly different (P>0.05). 35 d after the experiment, the serum IGF-1 (x±s, the same below) in the control group, mild hypoxia and severe hypoxia were (60.0±18.5) ng/ml, (40.6±9.9) ng/ml and (13.1±8.6) ng/ml, F=25.840, P<0.01; the serum IGFBP-3 were (1.93±0.23) µg/ml, (1.39±0.30) µg/ml and (0.90±0.21) µg/ml, F=33.929, P<0.01. The differences were statistically significant. The IGF-1 and IGFBP-3 levels decreased as the hypoxia increased (P<0.05).

CONCLUSIONIn simulated sleep apnea hypopnea syndrome, the intermittent hypoxia in young rats does not show physical growth retardation, but the serum IGF-1, IGFBP-3 levels decreased with the increase of hypoxia and decline of oxygen saturation.

Animals ; Disease Models, Animal ; Female ; Hypoxia ; blood ; Insulin-Like Growth Factor Binding Protein 3 ; blood ; Insulin-Like Growth Factor I ; metabolism ; Rats ; Sleep Apnea Syndromes ; blood

Pseudomonas sp. M18 is one of plant growth-promoting rhizobacteria capable of producing two kinds of anti-fungal agents: phenazine-1-carboxilic acid (PCA) and pyoluteorin (Plt). The pqsR gene, which encodes a LysR family member PqsR, was amplified from chromosomal genome of strain M18. Using the homologous recombination technique, a chromosomal pqsR inactivated mutant strain M18PRG was constructed in Pseudomonas sp. M18. To study the effect of pqsR gene on Plt biosynthesis, the dynamic curves of Plt production by strains M18 and M18PRG was measured in KMB media. As a result, Plt production of the pqsR mutant was three to four folds higher than that of its parent strain M18. The Plt production was restored to the wild-type level when strain M18PRG was complemented with pqsR gene in trans. The regulation of pqsR gene on Plt production was further confirmed by the pltA′-′lacZ translational fusion analysis. These results indicate that pqsR gene negatively controls the Plt biosynthesis. Additionally, by analyzing the growth curves of wild type strain M18 and pqsR mutant, wecan readily find that PqsR has a negative influence on cell growth. It was also shown that the production of red pigments in strain M18 required the expression of pqsR gene. In conclusion, the data presented in this study clearly demonstrate that PqsR acts as a global regulator involved in many physiological activities in Pseudomonas sp. M18.

OBJECTIVETo explore the myelo-protection effect of mdr1 transfected cord blood cells (CBMNCs) graft against high-dose homoharringtonine leukemia-bearing severe combined immunodeficient (SCID) mice model.

METHODSMultidrug resistant (mdr1)gene was transferred into CBMNCs by a retrovirus vector, containing full-length cDNA of human mdr1 gene. CBMNCs and high-titer retrovirus supernatant were cocultured with cytokine combinations for 5 - 6 days. The SCID mouse models bearing human HL-60 cell leukemia were divided into three groups. Group A received tail vein injection of 2 x 10(6) mdr1 gene transduced CBMNCs at day 1 and 3, groups B and C 2 x 10(6) un-transduced CBMNCs and same volume of normal saline, respectively. The 3 groups of the mouse model were treated with weekly escalated doses of homoharringtonine. The peripheral white blood cell (WBC) counts, the human leukemia cells percentage in peripheral blood, the histological findings of main organs were assayed. The CD33 positive HL-60 cells in bone marrow were determined by flow cytometry. The function and expression of mdr1 gene were examined by PCR, immunochemistry (IC) and DNR extrusion test in vivo.

RESULTS(1) mdr1 gene was transferred into CBMNCs successfully and the transfection frequency was 30%. (2) Leukemia SCID mice were xenotransplanted with mdr1-transfected BMMNCs by a programmed procedure and could be used as a valuable model for in vivo evaluating myelo-protection effects. (3) The transfected mice could tolerate homoharringtonine 5 approximately 6 folds higher than conventional dose and kept peripheral WBC count at a mean of 3 x 10(9)/L, with the peripheral human myeloid leukemia cells percentage decreasing to less than 5%. Histological examination showed that there was no leukemia infiltration in the main organs, the CD33 positive HL-60 cells in bone marrow were less than 5%. (4) The repopulation frequency of the transfected CBMNs in marrow were 9.13%. DNR extrusion test confirmed that the P-gp product maintained its biological function in the marrow.

CONCLUSIONmdr1 transferred-human CBMNC can xenotransplanted and repopulated in leukemia-bearing SCID mouse and are protected from chemotherapy-induced myelosuppression.

ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; metabolism ; Animals ; Antineoplastic Agents, Phytogenic ; administration & dosage ; adverse effects ; therapeutic use ; Cord Blood Stem Cell Transplantation ; methods ; Female ; Fetal Blood ; cytology ; Genetic Vectors ; HL-60 Cells ; Harringtonines ; administration & dosage ; adverse effects ; therapeutic use ; Humans ; Leukemia, Promyelocytic, Acute ; drug therapy ; pathology ; surgery ; Leukocytes, Mononuclear ; cytology ; metabolism ; transplantation ; Male ; Mice ; Mice, SCID ; Random Allocation ; Retroviridae ; genetics ; Transfection ; Treatment Outcome ; Xenograft Model Antitumor Assays

OBJECTIVETo observe the long-term results of midline partial glossectomy with uvulopalatopharyngoplasty (UPPP) in the treatment of obstructive sleep apnea hypopnea syndrome (OSAHS).

METHODSTwenty-four severe OSAHS patients treated with midline partial glossectomy and UPPP from January 2003 to March 2004 were included in this study, the follow-up was 5 years. The median of preoperative lowest arterial oxygen saturation (LSaO(2)) of this group at night (the same below) 0.650, and AHI was 56.5 times/h, UPPP was performed under general anesthesia, no tracheotomy performed. Criteria of curative effects: AHI < 5 times/h was recovery, AHI < 20 times/h and decreased beyond 50% marked improvement, only AHI decreased beyond 50% improvement.

RESULTSPost-operation AHI (6 months, 1 year, 2 years and 5 years after surgery) decreased significantly compared to that before the surgery, and post-operation LSaO(2) was significantly higher than that of preoperative (Wilcoxon's signed rank test, the same below, P < 0.01). The LSaO(2) and AHI were significantly different between 1 year, 2 years, 5 years and 6 months post-operatively (P < 0.01). Six months after surgery, PSG results showed that 21 were recovery, marked improvement for the other 3 cases, the recovery rate was 87.5%. One year after surgery, 18 were recovery, marked improvement in 3 cases, the recovery rate 75.0%. Two years after surgery, 14 cases recovery, marked improvement in 4 cases, the recovery rate 58.3%. Five years after surgery, 6 were recovery, the recovery rate 25.0%. Among 5 cases with hypertension before the surgery, after surgery antihypertensive drugs were not necessary in 4 cases, and the dosage was decreased in 1 case.

CONCLUSIONThe midline partial glossectomy with UPPP surgery may be an effective treatment for the severe OSAHS, long-term effect is satisfactory.

Adult ; Follow-Up Studies ; Humans ; Male ; Middle Aged ; Otorhinolaryngologic Surgical Procedures ; methods ; Palate, Soft ; surgery ; Retrospective Studies ; Sleep Apnea, Obstructive ; surgery ; Tongue ; surgery ; Uvula ; surgery

OBJECTIVETo establish a novel Myc gene transgenic mouse model for spontaneously forming B-lymphoma and assessing its tumorigenesis potential.

METHODSFreshly isolated hematopoietic progenitor cells served as the target for Myc gene transfer mediated by a retrovirus vector. These cells were engrafted into C57BL/6 mice with (60)Co-gamma ray radiation in advance. Tumor latency was measured and the tumor loaded mice were followed for survival time. Tumor was identified with histology and immunostaining. The exogenous Myc gene was detected by Western blot (in liver, spleen, tumor tissue) and flow cytometry (FCM) \[in bone marrow (BM)\].

RESULTSMice BM-infected with mutant Myc gene more readily gave rise to B-cell lymphomas than those infected with wild type Myc gene did Myc gene was expressed highly in BM and tumor tissues but not in liver and spleen.

CONCLUSIONOur model will be a tool in assessing the transforming potential of Myc mutants and in studying cooperation between Myc and other oncogenes. Mutant Myc is more effective than wild-type Myc in promoting B cell lymphomagenesis in mice.

Animals ; B-Lymphocytes ; Cell Transformation, Neoplastic ; Flow Cytometry ; Lymphoma ; Lymphoma, B-Cell ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Retroviridae Infections

OBJECTIVETo observe changes of growth, body composition and biochemical markers associated with growth (IGF-1) in prepubertal children with obstructive sleep apnea syndrome (OSAHS).

METHODSThirty-one children aged 3-10 years with OSAHS were followed up for 1 year after the corresponding surgery. During the same period of time, 20 children of similar age without OSAHS (excluded any other diseases that could result growth retardation or hypoxemia) were also followed up for 1 year. PSG, height, weight as well as insulin-like growth factor-1 (IGF-1) were measured during the preoperative period, 3 months, 6 months and 1 year after surgery in patient group. The same indexes were measured before surgery, and only height and weight were recorded after surgery in the control group. Wilcoxon signed- rank test and Mann-Whitney U test are used to analyze the data.

RESULTSThe lowest oxyhemoglobin saturation of the patient group (0.88) is significantly lower than that of the control group (0.98), and was found increased at the 6 months post-op follow up (0.97, U = 238.5, P > 0.05), no significant change was found at the 1 year follow up. The post-op AHI (6 months after surgery) of the patient group (6.0/h) decreased to the similar level of the control group (0/h, U = 240.0, P > 0.05), and was similar to 1 year after surgery. Height of the patient group (116 cm), which was lower than the control group (U = 127.0, P < 0.001), significantly increased 1 year (138 cm) after the corresponding surgery (Z = 3.726, P < 0.01), and reached the similar levels of the control group (137 cm) 1 year after the surgery (U = 123.5, P > 0.05). The serum IGF-1 levels of the patient group (33.7 ng/ml), which were significantly lower than those of the controls preoperatively (44.1 ng/ml, U = 206.0, P < 0.05), increased to similar levels with the controls 6 months after the operation (50.3 ng/ml, U = 261.0, P > 0.05), and the 1 year post-op follow up was similar to the control group too (48.6 ng/ml, U = 163.0, P > 0.05).

CONCLUSIONSThe cure of OSAHS could accelerate growth in prepubertal children, and the serum IGF-1 levels increases at the same time. The growth retardation is presumed in children with OSAHS.

Body Height ; Body Weight ; Case-Control Studies ; Child ; Child, Preschool ; Female ; Growth Disorders ; etiology ; Humans ; Insulin-Like Growth Factor I ; metabolism ; Male ; Sleep Apnea, Obstructive ; blood ; therapy ; Treatment Outcome

OBJECTIVETo explore the expression of connective tissue growth factor (CTGF) in pancreatic cancer and its influence on the proliferation and migration of cancer cells.

METHODSThe expression of CTGF in pancreatic cell line PANC-1 cells was analyzed by real-time PCR and in pancreatic carcinoma (50 cases) tissues by immunohistochemistry. The ability of proliferation and migration in vitro of PANC-1 cells was tested by MTT assay, scratch test and Boyden chamber test after the CTGF gene was overexpressed by Ad5-CTGF or silenced with Ad5-siCTGF transfection.

RESULTSCTGF was overexpressed in both pancreatic cancer cells and tissues. Overxpression of CTGF leads to increased proliferation and migration of PANC-1 cells. The CTGF-transfected PANC-1 cells showed apparent stronger proliferation ability and scratch-repair ability than that of empty vector controls. The results of Boyden chamber test showed that there were 34 cells/field (200× magnificantion) of the CTGF-transfected overexpressing cells, much more than the 11 cells/field of the empty vector control cells; and 6 cells/microscopic field of the Ad5-siCTGF-transfected silenced cells, much less than the 15 cells/field of the control cells.

CONCLUSIONSCTGF is overexpressed in both pancreatic cancer cells in vitro and in vivo, indicating that it may play an important role in the cell proliferation and migration in pancreatic cancer.

Adenoviridae ; genetics ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Connective Tissue Growth Factor ; genetics ; metabolism ; Humans ; Pancreatic Neoplasms ; metabolism ; pathology ; Recombinant Proteins ; genetics ; metabolism ; Transfection

OBJECTIVETo establish and evaluate a new method for measurement of dental plaque by using cellular neural network-based image segmentation.

METHODSA total of 195 subjects were selected from community population. After dental plaque staining, oral digital picture of anterior teeth area was taken by an Olympus digital camera (C-7070 Wide Zoom). At the same time, the Turesky dental plaque indices of anterior teeth were evaluated. The image analysis was conducted by cellular neural network-based image segmentation.

RESULTSThe image cutting errors between two operators were very small. The Kappa value is 0.935. Pearson's correlation coefficient is 0.988 (P < 0.001). There was high correlative consistency between traditional dental plaque index and plaque percentage obtained by using image analysis. Pearson's correlation coefficient was 0.853 (P < 0.001).

CONCLUSIONSCellular neural network-based image segmentation is a new method feasible for evaluating dental plaque.

Dental Plaque ; diagnosis ; Dental Plaque Index ; Female ; Humans ; Image Processing, Computer-Assisted ; Male ; Neural Networks (Computer) ; Photography, Dental

[Objective]To study the relationship between bridging septa thickening in the perinephric space and split renal function in acute unilateral upper urinary tract obstruction.[Methods]50 patients with acute unilateral upper urinary tract obstruction by calculus were analyzed retrospectively. According to the images of multi-slice spiral CT (MSCT)scanning,all cases were divided into thickened bridging septa group(n=25)and normal bridging septa group (n=25),The CT values of renal cortical in the plain(CTp)and renal cortical enhancing(CTe)phase were measured, the difference of increasement value(CTe-CTp)and the CT value ratio of the ipsilateral and opposite in renal cortical phase was analyzed by independent sample T test.[Results]The CT increasement value of bridging septa thickening and normal group were(103±30)HU and(128±24)HU respectively,the difference between the two groups was statistically significant(P<0.01);and CTac/CTuc were 0.81±0.13 and 0.96±0.06 respectively(P<0.01).[Conclusion]Thickening of bridging septa in the perinephric space with acute unilateral upper urinary tract obstruction will weaken the enhancement of renal cortical,and increased the likelihood of split renal function impairing.