The purpose of this paper is to investigate the reversal effect of small interfering RNA (siRNA) targeting MDR1 and MDR3 genes on the resistance of MCF-7/ADR cells to adriamycin. siRNA plasmid vector targeting MDR1 and MDR3 genes was transfected into MCF-7/ADR cells, and then was stained with Annexin-V FITC (fluorescein isothiocyanate conjugated) to detect the early stage cell apoptosis by flow cytometry (FCM). 50% inhibition concentration (IC50) of adriamycin for MCF-7/ADR cells was determined by MTT method. MDR1 and MDR3 mRNA was assessed by RT-PCR. Treatment of MCF-7/ADR cells with the two kinds of siRNAs resulted in a reversal of adriamycin resistance of MDR to different extents. 1) The apoptosis efficiency of MDR1 and MDR3 siRNA vector after transfection was (18.21+/-1.65) % and (9.07+/-2.16) % respectively (P<0.05), and there was significant differences in the apoptosis efficiency between pSuppressor Neo vector and the MDR1siRNA or MDR3 siRNA vector (P<0.01); 2) The reversal effect of MDR1 siRNA is higher than that of MDR3 siRNA (P<0.05); 3) The expression of MDRI and MDR3 mRNA can be restrained by pSuppressor Neo MDR1 and MDR3 siRNA respectively, and the reduction in the mRNA level was in a time-dependent manner (P<0.01). MDR1 and MDR3 gene silencing can enhance intracellular adriamycin accumulation in MCF-7/ADR cells, improve sensitivity of MCF-7/ADR cells to adriamycin, and induce cell apoptosis. The reversal effect of adriamycin resistance by siRNA of MDR1 was more effective than that of MDR3.

To explore the role and possible mechanism of apoptosis and caspase-3 activity in the development of multicellular drug resistance of ovary cancer. Ovarian cancer cell A2780 multicellular spheroids (MCS) were obtained from three-dimensional culture. Drug sensitivity of monolayer cells (MC) and MCS were respectively tested by MTT staining and cytometry. The apoptosis of MC and MCS were determined by the flow cytometry (FCM). The expression of bcl-2 and caspase-3 in A2780/MC and A2780/MCS were detected by using Western blot and caspase-3 assay kit. A2780/MC was compacted into mass after 2 days in three-dimensional cell culture model, and MCS had more than two layers of cells growing within 5 days. Compared with A2780/MC, A2780/MCS were more resistant to the anticancer drug, and the apoptosis rate was significantly lower than those of A2780/MC. The activity of caspase-3 in A2780/MCS was significantly lower than the A2780/MC. But the expression of bcl-2 in A2780/MCS was significantly higher than that in A2780/MC. It was suggested that the drug resistance of MCS might be associated with the overexpression of anti-apoptosis protein bcl-2 and the down-regulation of caspase-3 activity.

Voltage-gated sodium channels (VGSCs) are known to be involved in the initiation and progression of many malignancies, and the different subtypes of VGSCs play important roles in the metastasis cascade of many tumors. This study investigated the functional expression of Nav1.5 and its effect on invasion behavior of human breast cancer cell line MDA-MB-231. The mRNA and protein expression of Nav1.5 was detected by real time PCR, Western Blot and immunofluorescence. The effects of Nav1.5 on cell proliferation, migration and invasion were respectively assessed by MTT and Transwell. The effects of Nav1.5 on the secretion of matrix metalloproteases (MMPs) by MDA-MB-231 were analyzed by RT-PCR. The over-expressed Nav1.5 was present on the membrane of MDA-MB-231 cells. The invasion ability in vitro and the MMP-9 mRNA expression were respectively decreased to (47.82+/-0.53)% and (43.97+/-0.64)% (P<0.05) respectively in MDA-MB-231 cells treated with VGSCs specific inhibitor tetrodotoxin (TTX) by blocking Nav1.5 activity. It was concluded that Nav1.5 functional expression potentiated the invasive behavior of human breast cancer cell line MDA-MB-231 by increasing the secretion of MMP-9.

Objective:The different subtypes of voltagE-gated sodium channels (VGSCs) are known to correlate with the migration of many malignant cancers. This study was to investigate the significance of functional expression of SCN5A/Nav1.5 in human ovarian cancer and its effects on migration capability of ovarian cancer cells in vitro. Methods: Sodium indicator SBFI and immunofluorescence method were used to detect the distribution of intracellular Na+. Real-time PCR, Western blotting, and immunohistochemistry were used to detect the mRNA and protein expression of SCN5A/Nav1.5. The effect of specific voltagE-gated sodium channels inhibitor tetrodotoxin (TTX) on cell viability was measured by CCK-8 kit. The migration and invasion of ovarian cancer cell lines SKOV-3 were tested by Transwell chamber assay. Results:SCN5A/Nav1.5 were over-expressed in ovarian cancer cell lines SKOV-3 and ovarian cancer specimens at mRNA and protein levels. TTX 30 μmol/L inhibited the intracellular Na+ concentration by (41.51±0.41)%. TTX also suppressed the invasion and migration capacities of SKOV-3 cells by (33.80±1.6)% and (43.60±2.9)%, respectively. The difference was significant (P<0.05). Conclusion:SCN5A/Nav1.5 is involved in the metastasis progression of ovarian cancer in vitro and plays an important role in the initiation and progression of ovarian cancer. It may become a target for ovarian cancer therapy.

For treatment of pediatric inguinal hernia, we fabricated a device, i.e. so called "filling type pediatric hernia sac", which treats the problem from the abdominal cavity, through the abdominal and is a self-adaptive closer, using synthetic material. The device includes filling rack, self-adaptive umbrella support bar, bottom piece, outside pulling line and device fixing lines. The filling rack is composed of 2 concentric circles of 3.0 cm diameter with peripherally fixed together and can be pulled into the shapes of a ball or an olive. The supporting bar is structured of 3 pieces with 0.5 cm wide, 4.0 cm long, cross-fixed on top of the filling rack. The bottom piece is in a circular structure with a diameter of 3.0 cm, and it is connected to the filling rack bottom. Adjust positioning stay outside the fixed on the top of the device are connected at one end, and the other end free through filling the top frame connected with the bottom slice of central fixation. By using this device, we treated 37 pediatric inguinal hernia cases with 38 side-inguinal hernia successfully. The mean duration of post-operation follow-ups was 14.6 ± 5.89 months, without hernia recurrence, obvious scar and hard sections of inguinal region. This device could provide a convenient, safe and effective plugging technology for children's pediatric hernia.
Child ; Hernia ; therapy ; Herniorrhaphy ; instrumentation ; Humans

Objective To study the expressive changes of RAR-β gene in cervical carcinogenesis and analyze the relation between this differentiated expression and methylation, and discuss the clinical significance of this differentiated expression.Methods RT-PCR method was used to detect the expression of RAR-β in 4 cervical eancer cell lines,38 eases of cevical invasive carcinomatous tissues,30 cases of cevical intraepithelial neoplasia,and 10 cases of normal cervical tissues.Using Western Blot method to detect the expression of RAR-β protein quantificationally, analyze the RAR-β gene expressive difference in protein level.Using the methylation-*specific PCR methods to analyze the RAR-β methylation state in cell lines and tissues.Meanwhile analyze the relationship between the RAR-β gene expression and the clinical pathological parameters.Resuits The expression of RAR-β gene was deleted or deereaseed in cervieal cancer celllines,cevical intraepithelial neoplasia and cevical carcinoma,which was significantly difierent from normal tissues(P＜0.05).The same results was found in the protein expression.The methylation in RAR-β gene first exon was detected in 3 types of cell lines,11 cases of carcinoma,1 case of CIN Ⅰ and 5 cases of CIN Ⅱ～Ⅲ.while the RAR-β gene expression was decreased in those cell lines and tissues.There was significant differenee of RAR-β gene methylation in different pathological grades (P=0.037).There was no statistical significant difference of clinical stages(P≈1.000)and the metastasis of lymph node(P≈1.000).Conclusion The defective expression of RAR-β gene plays an important role in the carcinogenesis of cervical cancer.The abnormal methylation of promoter region was the main cause of defective expression of RAR-β gene.

Objective To observe the expression of RAR-β gene in SiHa, HeLa,C33A and CasKi cell lines of cervical carcinoma and to investigate the role of methylated RAR-β in its expressive defection. Methods Reverse transcription polymerase chain reaction (RT-PCR) was used to analyze the mRNA expression of RAR-β gene. Immunohistochemistry and Western Blot were used to analyze the protein expression of RAR-β gene in four cervical cancer cell lines as well as the influence of 5-Aza-cdR on gene expressive defection. Methylation specific PCR (MSP) was used to detect whether there was the methylation in RAR-β gene in four cell lines. The change of RAR-β gene methylation state was also observed by MSP. The cell proliferation rate influenced by the 5-Aza-cdR was observed by MTT assay. Results The expression of RAR-β mRNA and protein in SiHa, HeLa and CasKi cell lines of cervical cancer was silent or decreased, whereas its expression was detected in C33A cell line. By using MSP method, it was found that there was RAR-β gene methylation in those three cell lines, whereas there was no RAR-β gene methylation in C33A cell line. After treated with the 5-Aza-cdR, methylated RAR-β gene was partly demethylated, and RAR-β mRNA and protein were re-expressed in the previous three cell lines in which RAR-β gene expression was silent or decreased. The 5-Aza-cdR treatment could supress cell proliferation as well. Conclusion The RAR-β gene expressive defection plays an important role in the carcinogenesis of cervical cancer. The abnormal RAR-β gene methylation in the the promotor region has an important role in gene expressive defection. The cell proliferation can be supressed by demethylated treatment.

Objective To observe the effects of TLR9 on the nude mouse transplanted tumor growth of human pancreanc cancer and its drug resistance.Methods The nude mouse transplated tumor of human pancreatic cancer PANC-1 was established and randomly divided into 6 groups for conducting the experiment:sterile normal saline group,TLR9 agonist,TLR9 inhibitor group,gemcitabine group,TLR9 inhibitor plus gemcitabine Bin group,TLR9 agonist plus gemcitabine.The tumor size and growth situation were recorded by the vernier caliper.The immunohistochemical method was used to detect tumor TLR9 receptor expression.The tumor growth,metastasis and paracancerous nssue invasion situation were observed by the magnetic resonance imaging (MRI).Results The volume and growth speed of resected tumor mass in the gemcitabine group,TLR9 agonist + gemcitabine group,TLR9 inhibitor plus gemcitabine group was significantly smaller than those in other groups (P＜0.05),which in the TLR9 agonist + gemcitabine group were significantly greater than those in the TLR9 inhibitor plus gemcitabine group and gemcitabine group (P＜0.05),the difference between the TLR9 inhibitor plus gemcitabine group and gemcitabine group had statistical significance (P＜0.05),while the difference among the TLR9 agonist group,TLR9 inhibitor group and normal saline group had no stastistical significance (P＞0.05).The tumor in mice at 7 weeks after planting showed oval shape with clear boundary by MRI observation,no obvious metastais and paracancerous invasion were seen in paracancerous nssues no statistically significant,5 weeks,6 weeks after planting,seven weeks mice observed in MRI,the tumor into an,state clearly that the transfer of the surrounding tissue,no significant vascular invasion,heart,liver,kidney disease.The TLR9 expression on the surface of tumor tissue was detected and identified.Conclusion Pancreatic cancer nude mouse transplated tumor has definitely positive expression of TLR9,TLR9 activation can significantly decrease the sensitivity of pancreatic cancer to chemotherapy,increases the drug resistance of tumor,on contrary promotes the tumor growth.

OBJECTIVE: Poly (ADP-ribose) polymerase (PARP) is an important molecule in the early stress response of DNA damage, which is involved in DNA damage repair and cellular senescence. Olaparib, as PARP inhibitor, has an anti-tumor effect on high grade serous ovarian cancer, but its effects on cellular senescence have not been reported. This study intends to explore the role of olaparib in the regulation of senescence in ovarian cancer cells. METHODS: The effects of olaparib on the senescence of ovarian cancer cells were detected by using the senescence-associated β-galactosidase (SA-β-Gal) and senescence-associated heterochromatin aggregation (SAHF). Quantitative real-time polymerase chain reaction was used to analyze the senescence-associated secretory phenotype (SASP). Cell cycle and apoptosis were detected by flow cytometry. The effect of olaparib on tumor growth was analyzed in a nude mouse xenograft transplantation model. RESULTS: Long-term (6 days) treatment with olaparib (5 μM) significantly inhibited the growth of ovarian cancer cells, leading to arrest the cell cycle at G0/G1 phase, significant increase the number of positive SA-β-Gal stained cells and positive SAHF cells. The expression of P16 and retinoblastoma protein (p-RB) were significantly enhanced in SKOV3 cells under olaparib treated, meanwhile, the expression of P53 and p-RB were upregulated in A2780 cells. In OVCAR-3 cells, the expression of P53 was downregulated and p-RB was upregulated. Mice with SKOV3 xenograft transplantation was given olaparib (10 mg/kg/day) via abdominal cavity administration, the tumor volume was reduced (p < 0.01). CONCLUSION: Continuous low dosage administration of olaparib induced senescence under P16 or P53 dependent manner in ovarian cancer.
Abdominal Cavity ; Aging ; Animals ; Apoptosis ; Cell Aging ; Cell Cycle ; DNA Damage ; Flow Cytometry ; Heterochromatin ; Mice ; Mice, Nude ; Ovarian Neoplasms ; Phenotype ; Real-Time Polymerase Chain Reaction ; Retinoblastoma Protein ; Transplantation, Heterologous ; Tumor Burden

Objective To study the effect of silica in soil on the extraction of biological evidence DNA at the crime scene using the silica bead method.Methods Mud suspension and diluted blood were mixed to prepare biological samples mixed with dust and soil,which is to simulate biological evidence at the crime scene.Cell lysis was performed using heating lysis and guanidine salt chemical lysis,respectively.DNA was extracted using the silica bead method,amplified by PCR using Identifiler Plus kit and detected by capillary electrophoresis.The electrophoresis results were compared.Using mud suspension instead of silica beads to extract diluted blood DNA to validate the effect of silica in soil on the extraction of biological evidence DNA at crime scene using silica beads method.Results The complete STR loci were obtained after the extraction and amplification of 4 μL,20 μL dilute blood mixed with mud and lysed with heating cracking,whoes average peak heights arel 969.7±376.9 RFU and 9 706.7±349.8 RFU.For the 4 μL dilute blood mixed with mud guanidine salt chemical lysis,it cannot obtain complete STR loci after extraction and amplification.20 μL dilute blood mixed with mud guanidine salt was chemically cleaved and amplified to obtain complete STR loci with an average peak height of 1 899.8±801.3 RFU.After extraction and amplification by mud suspension instead of silica beads to extract 20 μL diluted blood DNA,complete STR loci were obtained.Conclusion Silicon dioxide in soil can bind to DNA in the presence of guanidine salts,leading to a decrease in the efficiency of recovering on-site biological evidence DNA using the silicon bead method.