This research explored the synergistic effects and the potential mechanisms of RCE-4 and various nonsteroidal anti-inflammatory drugs (NSAIDs) on the proliferation of cervical cancer Ca Ski cells. The MTT assay and CalcuSyn V2.0 software were used to detect cell proliferation and calculate the combination index (CI); the expression levels of various proteins were analyzed using Western blot assay; mitochondrial membrane potential (MMP) was assessed using JC-1 staining; acridine orange/ethidium bromide (AO/EB) double-fluorescence staining was used to detect the apoptosis of Ca Ski cells; a co-immunoprecipitation (Co-IP) assay was used to analyze the relative content of Bcl-2-Beclin 1 complex in Ca Ski cells. The results demonstrate that the combination of RCE-4 and NSAIDs increases the inhibition of Ca Ski cells compared to the single-RCE-4 group, and celecoxib provided the best synergistic effect among the four NSAIDs tested, with a CI of 0.32. The combination of RCE-4 and celecoxib significantly down-regulated the expression of cyclooxygenase-2 (COX-2) and nuclear transcription factor-κB (NF-κB), and promoted the expression of non-steroidal anti-inflammatory drugs activity gene-1 (NAG-1). In addition, autophagy induced by RCE-4 was markedly inhibited in combination with celecoxib, which was associated with down-regulation of the expression of microtubule-associated protein 3 (LC3)-II, Beclin 1, p62 and autophagy-related gene (ATG) 3/4B/5/7/14. RCE-4-induced apoptosis was significantly enhanced by altering the depolarization of mitochondrial membrane potential and the expression of B cell lymphoma-2 (Bcl-2), B cell lymphoma-xl (Bcl-xl), Bcl-2 associated X protein (Bax), Bcl-2/Bcl-xl-associated death promoter (Bad) and cleaved cysteinyl aspartate specific proteinase (cleaved-caspase) 3/7/9. Furthermore, the formation of the Bcl-2-Beclin 1 complex was significantly inhibited in Ca Ski cells treated with RCE-4 in combination with celecoxib. Taken together, this research shows that the combination of RCE-4 and celecoxib has a significant synergistic effect on the proliferation of Ca Ski cells by promoting apoptosis, inhibiting autophagy and disturbing the formation of the Bcl-2-Beclin 1 complex, which may be a novel strategy to increase the sensitivity of anti-cervical cancer drugs.

Objective To explore the influence of the lentiviral vectors-mediated mouse genetic engineering regulatory T cells (Treg) infused after allogeneic bone marrow transplantation (alloBMT) on graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effect in mice.Methods Lentivirus-mediated expression of Forkhead box P3 (Foxp3) transformed CD4 + CD25- T cells from Balb/c mice into engineered Tregs in vitro. An allo-BMT model of Balb/c→C57BL/6 mice was established. The recipients were given lethal X-ray total body irradiation before transplantation.Mice were randomly assigned into five groups and each group contained 10 recipients: (1) The recipients in radiation group were injected with 0.2 ml RPMI 1640; (2) The recipients in leukemia control group were injected with 5 × 106 donor bone marrow cells and 500 mouse T-cell leukemia/lymphoma cells (EL4 cells); (3) The recipients in transplantation control group were injected with 5 × 106 donor bone marrow cells and 5 × 106 splenocytes plus 500 EL4 cells; (4) The recipients in engineering Treg group were injected with 5 × 106 donor bone marrow cells, 5 × 106 splenocytes and 500 EL4 cells plus 5 × 106 genetic engineering Treg; (5) The recipients in empty vector control group were injected with 5 × 106 donor bone marrow cells, 5 × 106 splenocytes and 500 EL4 cells plus 5 × 106 empty vector-transduced CD4+ CD25- T cells. Survival time, clinical GVHD score or histopathological analysis (skin, liver and small intestine) were observed after allo-BMT. Chimerism of bone marrow cells from recipients survived for 60 days after transplantation was measured. Results The mean survival time in radiation group, leukemia control group, transplantation control group,engineering Treg group and empty vector control group was ( 10. 3 ± 1.5), (20. 7 ± 1.9), (26. 0 ±4.3), (49. 0 ± 17. 7) and (24. 4 ± 4. 1 ) days respectively. The survival time in engineering Treg group was significantly prolonged as compared with other groups as judged by the log-rank test (P＜0. 05).Histopathological analysis in several target organs (skin, liver and small intestine) confirmed the presence of severe GVHD in transplantation control group and empty vector control group. No histological signs of GVHD or leukemia were observed in recipients in engineering Treg group and clinical GVHD scores in this group were significantly decreased as compared with transplantation control group and empty vector control group. Conclusion Co-injection of genetic engineering Treg can efficiently prevent recipients from lethal GVHD without affecting GVL activity during allo-BMT in mice.

Objective To determine the conditioning regimen suitable for mice allogeneic hematopoietic stem cell transplantation (allo-HSCT).Methods Twelve BALB/c mice were randomly divided into 2 equal groups to undergo X-ray irradiation by linear accelerator at the dose of 7.0 Gy (pure X-ray group) or 60Co source irradiation at the dose of 7.0 Gy (pure γ-ray group).Thirty mice were randomly divided into 2 equal groups to undergo X-ray irradiation and then infusion of bone marrow from donor mice via caudal vein (X-ray + transplantation group) or γ-ray and then infusion of bone marrow via caudal vein (γ-ray + transplahtation group).3,5,7,10,15,20,and 30 d later peripheral blood samples were collected to calculate the number of white blood cells (WBCs) and detect the chimeric rates of lymphocytes by flow cytometry.5,10,and 20 d after irradiation 15 mice were killed with their lung,liver,small intestine,spleen,and femurs taken out to undergo pathological examination.Results The survival rates during the period 5-15 days of the γ-ray + transplantation group were all significantly higher than those of the X-ray + transplantation group.The pathological changes of organs of the X-ray +transplantation group were all more severe than those of the γ-ray + transplantation group.Since the fifth day after transplantation cells originating from the donor began to appear in the peripheral blood.The chimeric rate of the γ-ray + transplantation group 10 days after transplantation was (95.53± 2.57) %.The chimeric rates 5,10,and 20 days after transplantation of the γ-ray + transplantation group were all significantly higher than those of the X-ray + transplantation group (t = 15.263,3.256,P < 0.05).The WBC count of both irradiation groups decreased to the lowest level 5 d later and began to increase 10 days after transplantation and the WBC counts of the γ-ray + transplantation group 10 and 20 days aftertransplantation were both significantly higher than those of the X-ray + transplantation group (t = 3.624,6.695 ,P < 0.05).The chimeric rats of the peripheral lymphocytes 10 and 20 days after transplantation of the γ-ray + transplantation group were both significantly higher than those of the X-ray + transplantation group (t = 12.317,8.295,P < 0.05).The homogeneity rate of transplantation of the γ-ray +transplantation group was better than that of the X-ray + transplantation group.Conclusions As a conditioning regimen in allogeneic hematopoietic stem cell transplantation γ-ray irradiation causes milder injury and accelerated reconstitution of hematopoiesis and immunity,in comparison with X-ray irradiation.

BACKGROUND:Theex-vivo expanded autologous adipose-derived stem cels have the capability of multipotential differentiation and have a broad application prospect in the field of tissue engineering and regenerative medicine. OBJECTIVE:To observe the nasal mucosal structural repair and functional reconstruction usingex-vivo expanded autologous adipose-derived stem cels. METHODS:Ten patients with mucosal damage due to the physical or chemical factors were enroled, including six cases of mucosal scar and four cases of mucosal ulceration. Autologous adipose tissue was extracted forin vitro isolation, culture and expansion of adipose-derived stem cels. Before transplantation, quality safety testing was done. Al the patients were injected adipose-derived stem cels (1×107/cm2 0.1 cm mucosal tissue sample at 30 days before and after transplantation for hematoxylin-eosin staining, Masson ) at an interval of 15 days, totaly for three times. Nasal volume, minimum cross-sectional area, and mucociliary clearance function were determined at 30, 90, 150 days after the final injection. Three of 10 patients were selected to take a 0.1 cm× trichrome staining, and AB-PAS staining. RESULTS AND CONCLUSION:Clinical symptoms were aleviated in al patients undergoing transplantation of adipose-derived stem cels. Compared with the baseline data, the nasal volume and minimum cross-sectional area were both decreased at 30, 90, 150 days after transplantation (P < 0.05), and the mucociliary clearance function was improved but not significantly (P > 0.05). Compared with the baseline data, the inflammation of the nasal mucosa was significantly reduced, colagen fibers arranged neatly, the deposition was decreased, and mucin secreted from goblet cels was increased in the selected three patients at 30 days after cel transplantation. These findings indicate thatex-vivo expanded autologous adipose-derived stem cels can be used to reconstruct the nasal mucosal structure and its function.

Background Scarring of the filtering bleb is a main cause of filtering surgical failure in glaucoma.It has been reposed that tetrandrine could suppress the proliferation of cultured human fibroblast of Tenons capsule in vitro and thus has the potential effect to prevent scarring after the filtering surgery. Objective Present study was to investigate the anti-cicatricial effect of tetrandrine drug delivery system(Tet DDS)during filtration surgery. Methods Filtration surgery was performed in bilateral eyes of 18 New Zealand white rabbits.The Tet DDS with 0.3 mg Tet,0.2 mg Tet or free-Tet were implanted subcunjunctially during the surgery.The filtering blebs were scored in 1 day,4,7,10,14 days after referring to the corneal thickness and bleb range under the slit-lamp biomicroscopy.The morphology of filtering bleb was assessed by in vivo confocal microscopy in 7 and 14 days after operation.The filtering bleb specimen was prepared in 7 and 14 days for the histopathological examination. Results The filtering bleb scores in Tet DDS implantation groups were significantly higher than those in free-Tet DDS group from 4 days through 14 days after trabeculectomy(P＜0.01),and the scores showed a considerably increase in 0.3 mg Tet DDS group compared with 0.2 mg Tet DDS group from 7 days through 14 days after trabeculectomy(P＜0.05).The filtering blebs of Tet DDS implantation groups were found with distinct subepithelial cystic spaces under the light microscopy and in vivo confocal microscopy on the 7th day and 14th day after surgery.Compared with free-Tet DDS group,the numbers of subepithelial mierocysts were much more(P＜0.01)and the area of microcysts was larger(P＜0.01)in Tet DDS group.The filtering tissue presented with more subepithelial microcysts and larger microcysts range in 0.3 mg Tet DDS group than 0.2 mg Tet DDS group in 7 and 14 days after operation(P＜0.05).The inflammatory cell infiltration wag milder in 0.3 mg Tet DDS group in comparison with 0.2 mg Tet DDS group and free-Ted DDS group.Conclusion Tet DDS has strong inhibitory effects on inflammatory cells activity and fibroblagt activity the early stage after filtering surgery and therefore improve the surgery success rate.

Objective A prospective randomized controlled trial was carried out to explore the best time of In-domethacin suppositories administration for the prevention of post-ERCP pancreatitis in high-risk groups. Methods 81 patients were enrolled in the study finally. Patients were randomized into group A (100 mg rectal Indomethacin suppositories was administrated immediately after ERCP), group B (100 mg rectal Indomethacin suppositories was administrated half an hour after ERCP) and group C (ERCP alone group, which did not give Indomethacin supposito-ries). The level of serum amylase, urine amylase, serum CRP, serum IL-6, serum TNF-α were measured before, 3 h, 24 h and 48 h after ERCP, and the incidence of PEP and hyperamylasemia were analyzed. Results There was 1 case (4.00%) of PEP in group A, 2 cases (5.41%) of PEP in group B and 5 cases (26.31%) of PEP in group C;the incidences of PEP of group A and group B were significant lower than that in group C (P < 0.05). There was 1 case (4.00%) of hyperamylasemia in group A, 5 cases (13.51 %) of hyperamylasemia in group B and 6 cases (31.6 %) of hyperamylasemia in group C, and the incidences of hyperamylasemia of group A and group B were significant lower than that in group C ( P< 0.05). Conclusion Administration of 100 mg Indomethacin suppositories immediately or half an hour after ERCP can effectively reduce the incidence of PEP and hyperamylasemia.

Objective To investigate the frequencies of circulating dendritic cell (DC) subsets and the function of monocyte-derived dendritic cells in patients with hepatitis B-related acute-on-chronic liver failure.Methods Peripheral blood was collected from hepatitis B-related acute-on-chronic liver failure patients (ACLF,n =40) and chronic hepatitis B (CHB,n =40) as well as normal controls (NCs,n =20).Circulating myeloid dendritic cell (Mdc) and plasmic dendritic cell (pDC) frequencies in peripheral blood mononuclear cells (PBMC) were analyzed by flow cytometric analysis.Purified monocytes were isolated by combination of Histopaque-1.077 and CD14 Microbeads.Monocyte-derived dendritic cells (MoDCs) generated in vitro in the presence of interleukin (IL)-4 and granulocyte macrophage colony-stimulating factor upon activation by poly I:C.Costimulatory molecule expression and allostimulatory mixed lymphocyte reaction (AMLR) of MoDCs were detected in patients with hepatitis B-related ACLF.Results The number of circulating mDC decreased only in patients with hepatitis B-related ACLF compared with that in normal controls.However,pDC numbers decreased in both CHB and ACLF patients.We observed a further decrease the pDC numbers in ACLF compared to CHB patients without statistical significance (P ＞ 0.05).MoDC from ACLF patients showed lower expression of costimulatory molecules CD80,CD86 and the mature marker CD83,as well as MHC Ⅱ molecule (HLA-DR) compared to CHB and NC group.Interestingly,MoDC impaired allostimulatory mixed lymphocyte reaction from ACLF patients compared to those in CHB patients and NCs.Conclusions Patients with hepatitis B-related ACLF have a significantly lower expression of surface markers and impaired AMLR of MoDC,as well as decreased number of circulating mDC and pDC,which may be partially related to HBV disease progression in these patients.

Objective To explore the incidence and risk factors of acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Methods The clinical data of 72 cases allo-HSCT from Oct 2004 to Dec 2008 were analyzed. Thirteen factors possibly correlated with the development of aGVHD were analyzed. Results aGVHD was developed in 32 cases (44.4 %), in which grades Ⅰ aGVHD was 11.1%, gradesⅡaGVHD was 18.1%, and grades Ⅲ-Ⅳ aGVHD was 15.3 %. The univariate analysis showed that diagnosis, the status of disease, use ATG, conditioning regimen, donor type,ABO blood group disparity between donor and recipient, CD34+ cell number, early engraftment and neutropenic infection, HLA locus were associated with the occurence of aGVHD (P ＜0.1). On the COX regression mode, an increased risk of aGVHD was associated with HLA mismatch (HR =2.58, P ＜0.005), GVHD prophylaxis without ATG (HR =2.94, P ＜ 0.001), and unrelated donor (HR =1.97, P ＜0.01). Conclusion aGVHD is a common complication after allo-HSCT, and HLA mismatch and unrelated donor are independent risk factors for aGVHD.

Objective To investigate the imaging features of sarcomatoid carcinoma(SC)of lung on chest radiography and CT and to improve knowledge of SC.Methods The chest plain films,CT images and clinical data of all 7 cases of pathologically proved lung SC were retrospectively analyzed.Results All cases presented with a spheroid solid lung mass ranged from 3 to 11 cm in diameter(average 7 cm).All the lesions were located in middle and lower fields of the lung:3 lesions in right lower lobe,2 in right middle lobe,and the other 2 in left lower lobe.Among the 7 cases:6 cases were of peripheral type and 1 case was of central type.The peripheral-typed lesions of 5 cases had clear margin and 3 of them were lobulated.Three massess were homogenous in density,and the other 4 were inhomogeneous in density with formation of central cavity or calcification.After intravenous contrast administration.Three lesions showed homogeneous enhancement,and the other 4 lesions had inhomogeneous enhancement.The lesions with diameter larger than 6 cm showed marginal or patch-like enhancement.Five cases had pleural or chest walls invasion.Two cases had mediastinal lymph nodes metastases.One case had multiple remote metastasis.Conclusion There are some relatively specific features of lung SC on chest radiography and CT,which may be helpful for diagnosis.

OBJECTIVE: To analyze and discuss four methods of calculating likelihood ratio of DNA mixture. METHODS: In the case with CNAS-T0757 proficiency testing in 2013, the likelihood ratios were calculated and compared among four methods, including unrestricted combinatorial method, Clayton's method, p2 principle method, and recommendations from ISFG. RESULTS: The likelihood ratios were maximum by Clayton's method and recommendations from ISFO, followed by result of the unrestricted combinational method. The minimum likelihood ratio was obtained by p2 principle. CONCLUSION The unrestricted combinational method could give fUrthest consideration to both information preservation and appraiser protection.
DNA/genetics* ; DNA Fingerprinting ; Humans ; Likelihood Functions