Transglutaminase 2 (TGM2) is a ubiquitous multifunctional protein, which is related to the adhesion of different cells and tumor formation. Previous studies found that TGM2 is involved in the interaction between host cells and viruses, but the effect of TGM2 on the proliferation of influenza virus in cells has not been reported. To explore the effect of TGM2 during H1N1 subtype influenza virus infection, a stable MDCK cell line with TGM2 overexpression and a knockout cell line were constructed. The mRNA and protein expression levels of NP and NS1 as well as the virus titer were measured at 48 hours after pot-infection with H1N1 subtype influenza virus. The results showed that overexpression of TGM2 effectively inhibited the expression of NP and NS1 genes of H1N1 subtype influenza virus, while knockout of TGM2 up-regulated the expression of the NP and NS1 genes, and the expression of the NP at protein level was consistent with that at mRNA level. Virus proliferation curve showed that the titer of H1N1 subtype influenza virus decreased significantly upon TGM2 overexpression. On the contrary, the virus titer in TGM2 knockout cells reached the peak at 48 h, which further proved that TGM2 was involved in the inhibition of H1N1 subtype influenza virus proliferation in MDCK cells. By analyzing the expression of genes downstream of influenza virus response signaling pathway, we found that TGM2 may inhibit the proliferation of H1N1 subtype influenza virus by promoting the activation of JAK-STAT molecular pathway and inhibiting RIG-1 signaling pathway. The above findings are of great significance for revealing the mechanism underlying the interactions between host cells and virus and establishing a genetically engineering cell line for high-yield influenza vaccine production of influenza virus.
Animals ; Cell Proliferation ; Dogs ; Humans ; Influenza A Virus, H1N1 Subtype/genetics* ; Influenza, Human ; Madin Darby Canine Kidney Cells ; Protein Glutamine gamma Glutamyltransferase 2

Objective:To explore the initial experience of thoracoscopic repair with simplified mattress sutures in the treatment of diaphragmatic hernia in neonates without posterolateral rim of diaphragm.Methods:A retrospective review of the new simplified technique in 10 cases from March 2015 to October 2017 was performed.Of the patients, 6 cases were male, 4 cases were female.The age was 10min-1d, 7 cases were term newborns, and 3 cases were premature.The mean weight was 2.88 kg(ranged 2.3-3.5kg). All the 4 cases were left-sided.Two to three primary suture sites were taken from the relative intercostal region of the body surface projection of the defect.A snip incision about 1 mm of the skin was done.Two 2-0 non-absorbable sutures round the rib were inserted between the front edged of the defect and the diaphragm muscle through a syringe needle.The first thread was brought out of the body by the ring of the second thread and knot tying was made extracorporally.The posterolateral defect was closed; the knot was under the skin of intercostals space.Results:Ten neonates with CDH were repaired successfully using this new simplified technique.The mean operative time was 37.5min(ranged 25-60min) for each CDH repair.No cases required conversion to open surgery, blood loss was minimal.The 10 cases were followed up for 16.5 months(ranged 5-24 months), with no death and no recurrence.One neonate complicated with subcutaneous emphysema postoperatively and healed in one week.Conclusion:The new technique of thoracoscopic repairing with simplified mattress sutures when no posterolateral rim of diaphragm exists has all the advantages of thoracoscopy in neonates combined with the advantages of reduced operative time, simplicity, feasibility and definite curative effect and has the value of clinical popularization.

Objective To explore the clinical value of portal exposure in laparoscopic treatment of children with type Ⅲ biliary atresia (BA).Methods From June 2013 to October 2017,30 infants with type Ⅲ BA who treated with laparoscopic portoenterostomy in Huai'an Women and Children's Hospital were selected.A percutaneous suture was used to snare the round ligament and retract the liver,other percutaneous stay sutures were then introduced and fundus and neck of gallbladder were sutured to elevate the liver to expose the portal hepatis.The fibro cord and hepatic vessels were mobilized,and then two rubber bands were put around the left and right portal veins and hepatic arteries.The portal hepatis was exposed by laterally stretching the two elastic rubber bands.The fibro cord was removed and then laparoscopic portoenterostomy was accomplished.In 20 cases,the liver was enlarged,part of hepatic lobus quadratus was removed laparoscopiclly for exposure of the portal hepatis.Results There were 30 cases in this group,2 cases were converted to open surgery by a micro transverse incision.There was no surgical death.Time of laparoscopic procedure varied from 210 to 280 min.All cases survived the surgery without any intraoperative complications.Blood loss during operation was minimal,without necessity for blood transfusion.One case died of respiratory failure one week after surgery.Two cases were lost follow-up.Twenty-five cases were followed up for 3～51 months(mean 22.4 months).Three cases died because of repeated cholangitis and liver failure at 10,16,35 months postoperatively.Nineteen patients' total bilirubin had dropped to normal,three others' bilirubin levels dropped significantly after surgery.Conclusion The technique of laparoscopic hepatic porta exposure can help to complete hepatic portoenterostomy successfully,reduce the conversion rate of laparoscopic surgery,and improve the surgical effect.

Objective:To investigate the effects of different doses and infusion methods of recombinant mouse interleukin-18(rmIL-18) on the survival time and tumor diameter of the mice with hepatocellular carcinoma,and to elucidate the rational application of rmIL-18 in vivo.Methods:A total of 60 Babl/C mice were randomly divided into 5 μg rmIL-18 intraperitoneal injection group,0.5 μg rmIL-18 intraperitoneal injection group,0.5 μg rmIL-18 tumor injection group,cytotoxic T lymphocyte(CTL) intraperitoneal injection group,CTL tumor injection group and saline control group;there were 10 mice in each group.From the 10th day of inoculation,the mice in different rmIL-18 groups were injected with the corresponding doses and methods.The mice in different CTL groups were injected with tumor-specific CTL (1×106/mouse) by intraperitoneal and intratumoral injection.The mice in saline control group were injected with an equal volume (100 μL) of saline,the injections were performed 10 times.The diameters of mice were measured weekly and the survival time was recorded.Results:Compared with 5 μg rmIL-18 intraperitoneal injection group,0.5 μg rmIL-18 intraperitoneal injection group and saline control group,the tumor growth rate of the mice in 0.5 μg rmIL-18 tumor injection group was decreased (P<0.01)and the survival rate of the mice was increased (P<0.01);compared with 0.5 μg rmIL-18 intraperitoneal injection group,the tumor growth rate and the survival rate of the mice in CTL intraperitoneal injection group were decreased (P<0.01);compared with 0.5 μg rmIL-18 tumor injection group,the tumor growth rate and the survival rate of the mice in CTL tumor injection group were decreased (P<0.01).Conclusion:The best way for rmIL-18 anti-tumor effect is tumor injection and the effect has a dose-dependent manner.

Objective:To observe the specific killing effect on tumor cells of the spleen cells in mice immunized with three tandem repeats of CEA minigene DNA vaccine pcDNA-triCEA625-667 and to evaluate the safety of the vaccine. Methods: The BALB/c mice were randomly divided into blank vector group ( pcDNA3. 0 ) , haploid vaccine group ( pcDNA-CEA625-667 ) and tandem repeats vaccine group (pcDNA-triCEA625-667). The mice received a total of 4 intramuscular immunization every 10 days once. The changes of body weight,survival state were recorded and the levels of serum ALT and serum creatinine were detected. The specific CTL killing activity of spleen cells in accinated mice on mouse hepatoma cells(H22-CEA+),gastric cancer cells(MFC-CEA+),colorectal cancer cells ( CT26-CEA+) with high expression of CEA and mouse hepatoma cells ( H22-CEA-) without expression of CEA was detected. Results:The two vaccines had strong killing activity on CEA positive liver cancer,gastric cancer and colon cancer cells,and the difference was statistically significant ( P<0. 01 ) compared with the PcDNA3. 0 group. And they had almost no effect on CEA negative tumor cells (H22-CEA-). The killing activity on liver cancer cell(H22-CEA+) and gastric cancer cell(MFC-CEA+) induced by pcDNA-triCEA625-667 was stronger than that induced by pcDNA-triCEA625-667(P<0. 05). The survival status,change of body weight and function of liver and kidney of the mice were not affected by the vaccine. Conclusion:There was no adverse reaction in the course of vaccine immunization. The minigene DNA vaccine derived from CEA can induce tumor specific CTL effect and the immune response level elicited by three tandem repeats of minigene DNA vaccine was superior to that elicited by haploid vaccine.

Objective:To observe the inhibitory effect of haploid vaccine pcDNA-CEA625-667 and three tandem repeats of minigene DNA vaccine pcDNA-triCEA625-667 derived from CEA gene on tumor in mice bearing tumor and the changes of survival time. Methods:The experimental animal model of mouse liver cell carcinoma was established and the mice were immunized with pcDNA-CEA625-667 and three series of DNA vaccine. Some of the mice were treated with normal saline as control group. The growth curve of tumor growth curve was recorded and the effect of vaccine on the survival time of tumor bearing mice was observed. Results:Compared with the normal saline control group,the two vaccines were able to significantly inhibit the tumor size and growth rate ( P<0. 01 ) of CEA positive tumor bearing mice,the inhibition of pcDNA-triCEA625-667 vaccine group was significantly better than the pcDNA-CEA625-667 vaccine group (P<0. 01),while the two were not inhibited tumor growth in CEA negative tumor bearing mice. The average survival time of the pcDNA-CEA625-667 vaccine group was(48. 50±6. 73)d,and there was significant difference (P<0. 01) compared with the saline control group ( 39. 00 ± 6. 64 ) d. The survival time ( 48. 50 ± 6. 73 ) d of the pcDNA-triCEA625-667 vaccine group was significantly higher than that of the normal saline control group and the pcDNA-CEA625-667 vaccine group (P<0. 01). The survival time of CEA negative tumor bearing mice could not be prolonged in the two groups. Conclusion:Either the haploid or the three series of the DNA vaccine,were able to significantly inhibit tumor growth rate (P<0. 01) and significantly prolong the survival time (P<0. 01) of CEA positive tumor bearing mice,but they had no therapeutic effect on CEA negative tumor bearing mice.

Objective:To observe the immunological activity of haploid vaccine and three tandem repeats of minigene DNA vac -cine derived from Carcinoembryonic Antigen (CEA) gene.Methods:The immunoreaction was induced by intramuscular injection with pc-DNA3.0,pcDNA-CEA625-667 and pcDNA-triCEA625-667 in BALB/c.Four weeks after injection,the spleen cells and serum were separa-ted respectively from the mice for the in vitro assessment .Changes of the T lymphocytes subset was analyzed by flow cytometry .Lymph proliferation responses were tested by 3 H-TdR incorporation ,IFN-γ,IL-4 and GM-CSF in their cultural supernatants were detected with ELISA and seral IgG antibody against CEA were detected with Western blot and ELISA .Results:The difference of the ratio of CD 4+/CD8+of the mice immuned by pc-DNA3.0,pcDNA-CEA625-667 or pcDNA-triCEA625-667 was not significant.Lymph proliferation responses were more significant in the mice immuned by pcDNA-CEA625-667 and pcDNA-triCEA625-667 in a shorter time by contrast with na ?ve mice.Low tilter IgG antibody against CEA was detected in the antiserum of the mice immuned by repeats of minigene DNA vaccine , which suggested the activation of helper T-cell.ELISA showed that the level of IFNγin the 3 days culture of the splenocytes was rela-tively higher in the groups of minigene DNA vaccination than in the control groups ,while IL-4 expression was absent in all groups .The immune response level elicited by three tandem repeats of minigene DNA vaccine pcDNA -triCEA625-667 was superior to that elicited by pcDNA-CEA625-667 ,which showed that any immunogenic inadequacies in minigene presentation can be rectified by linking itself in a string-of-beads vaccine.Conclusion:The haploid vaccine and three tandem repeats of minigene DNA vaccine derived from CEA geneboth can not change the ratio of CD 4+/CD8+but can induce the activation of helper T-cell and skew T -cells toward Th -1 response.The immune response level elicited by three tandem repeats of minigene DNA vaccine was superior to thatelicited by haploid vaccine .

Objective:To research rmIL-18 in vitro culture system CCs induce tumor-specific cytotoxic T lymphocytes CTL and anti-tumor effect in mice.Methods:Used Stem SepTM immune magnetic cells separation method to culture mouse spleen NK cells,T cells and DCs,established culture systems in vitro;used of different approaches,different doses rmIL-18 to immunize HCC tumor-bearing mice,researched the effect of rmIL-18 on tumor growth rate and survival time.Results:rmIL-18 could induce and promote tumor-specific CTL-mediated killing effects in vitro culture system;tumor-specific CTL could significantly inhibit tumor growth(P<0.01) of and prolong the survival time of liver cancer tumor-bearing mice(P<0.01),and the effect was increased with rmIL-18 concentration increased(P<0.01),and intratumoral injection was superior to intraperitoneal injection(P<0.01).Conclusion:rmIL-18 can induce tumor-specific CTL in vitro and play a role in anti-liver cancer in mice.

Objective:To probe the expression of miR-126 and VEGF in breast cancer, and the anti-tumor effect of miR-126. Methods:The expression of miR-126 and VEGF in breast cancer tissues and cells were detected by qRT-PCR and Western blot;after transfection with miR-126 mimics into MDA-MB-231,expression of VEGF was detected again,MTT assay and cell scratch test were used to verify the influence of miR-126 on proliferation and migration of tumor cells. Results: The expression of miR-126 was lower in the breast cancer tissues and cells,the expression of VEGF was negative correlation with it,increasing the expression of miR-126 may decrease the expression of VEGF and inhibit the proliferation and migration of breast cancer cells. Conclusion:miR-126 can reduce the proliferation and migration of breast cancer cells by inhibiting the expression of VEGF,which play an anti-tumor effect.

Objective:To investigate the expression of microRNA-199a/b-3p (miR-199a/b-3p) in hepatocellular carcinoma ( HCC) tissues,and to explore the relationship with clinical outcomes. Methods: Real time quantitative PCR technique was used to measure the expression of miRNA-199a/b-3p in HCC tissues. The correlation between miR-199a/b-3p expression and the clinic pathological features of patients were analyzed. Results: Comparing with adjacent control, miRNA-199a/b-3p presented lower expressions in HCC tissues (P<0. 05);lower miR-199a/b-3p was found correlated with metastasis and poor survival. Conclusion:MiR-199a/b-3p take a crucial role in HCC metastasis and recurrence.