【Objective】 To investigate the relationship between hepatitis B virus X （HBx） protein and EGFR promoter， and the role of HBx protein in activating EGFR/PI3K/p-Akt signaling pathway and inhibiting apoptosis. 【Methods】 EGFR promoter plasmids were constructed and the relationship between HBx and EGFR promoters was characterized using a luciferase reporter assay. EGFR-overexpressing trophoblast cells were constructed， and EGFR expression in the overexpressing cells was knocked down using EGFR shRNA. The expression and localization of EGFR/PI3K/p-Akt were detected by Western blotting and confocal laser microscopy. Cell apoptosis was analyzed using flow cytometry. HBV plasmids carrying either full-length HBx or HBx with a deletion mutation （ΔHBx） and HBx plasmids were transfected into two types of trophoblast cells； HBx and PI3K/p-Akt protein expressions were detected by Western blotting. Cell apoptosis was analyzed using flow cytometry. 【Results】 Co-transfection of HBx and EGFR promoter plasmids in JEG-3 and HTR-8/Svneo cells significantly elevated the expression of EGFR promoter driven luciferase compared with the control group （P<0.01）. In EGFR-overexpressing cells， the expression of PI3K/p-Akt was significantly increased （P<0.01）， whereas the apoptosis rate was significantly decreased for JEG-3 cells and HTR-8/Svneo cells （both P<0.01）. These results were reversed in the EGFR-knock down group. When the intracellular HBx protein was expressed in JEG-3 and HTR-8 cells， PI3K/p-Akt protein expression was significantly increased （both P<0.05）， and the proportion of apoptosis was significantly decreased （both P<0.05）. 【Conclusion】 In placental trophoblast cells， HBx protein activates the expression of EGFR by acting on the EGFR promoter， and inhibits the apoptosis of trophoblast cells via the downstream EGFR/PI3K/p-Akt signaling pathway.

Cas1-and-Cas2-mediated new spacer acquisition is an essential process for bacterial adaptive immunity. The process is critical for the ecology of the oral microflora and oral health. Although molecular mechanisms for spacer acquisition are known, it has never been established if this process is associated with the morphological changes of bacteria. In this study, we demonstrated a novel Cas2-induced filamentation phenotype in E. coli that was regulated by co-expression of the Cas1 protein. A 30 amino acid motif at the carboxyl terminus of Cas2 is necessary for this function. By imaging analysis, we provided evidence to argue that Cas-induced filamentation is a step coupled with new spacer acquisition during which filaments are characterised by polyploidy with asymmetric cell division. This work may open new opportunities to investigate the adaptive immune response and microbial balance for oral health.

Objective: To evaluate the anti-tumor activity of mouse multi-subtype heat shock protein/peptide (mHSP/P) vaccine in combination with a programmed death ligand 1 (PD-L1) inhibitor in mouse sarcoma. Methods: Immunohistochemical staining and en-zyme-linked immunosorbent assay (Elisa) was used to quantitatively identify the expression of heat shock proteins (HSP70, HSP90, Grp94) in the sarcoma cell line MCA207. From the protein suspension prepared, mHSP/P and Grp94/peptide (Grp94/P) sarcoma vac-cines were isolated using chromatography and were identified by Western blot (WB). Flow cytometry was used to determine their cy-totoxic effects. The levels of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) produced upon mHSP/P and Grp94/P stimulation were measured by Elisa. The effect of sarcoma vaccines on the growth and survival of sarcoma was evaluated in mice. The expression of PD-L1 on the surface of MCA207 sarcoma cells was evaluated by immunofluorescent staining. The effect of IFN-γ treatment on the expression of PD-L1 was determined by WB. Animal experiments explored the effects of PD-L1 inhibitor in combination with mHSP/P treatment on tumors. Results: Tumor tissue carries a variety of HSP subtypes (HSP70, HSP90, Grp94). We successfully isolated sarco-ma tissue-derived mHSP/P and Grp94/P tumor vaccines, which were identified by WB; flow cytometry analysis demonstrated their cy-totoxicity. The levels of IFN-γ and TNF-α cytokines upon mHSP/P stimulation were significantly higher than that observed upon Grp94/P stimulation (P<0.05). The expression of PD-L1 on the surface of sarcoma cells increased with IFN-γ treatment. Animal experiments demonstrated that PD-L1 inhibitor in combination with mHSP/P significantly increased the immune response against tumor (P<0.05). Conclusions: Tumor-derived mHSP/P and Grp94/P can be used as tumor vaccines in animal models. The mHSP/P can elicit a stronger anti-tumor immune response than Grp94/P. IFN-γ stimulates the expression of PD-L1 in sarcoma cells, which results in immune eva-sion. The PD-L1 inhibitor in combination with mHSP/P increased the anti-tumor effect in the tumor microenvironment.

Objective To observe the effect of systemic chemotherapy on conditions of tumor infiltrating,metastasis and disease-specific survival (DSS) for advanced retinoblastoma (RB).Methods Forty-one patients with advanced RB who received enucleation were enrolled in this study.There were 26 males and 15 females,age at diagnosis was ranged from 2 to 72 months,with a mean of 23.08 months.There were 16 bilateral patients and 25 unilateral patients;13 group D eyes and 28 group E eyes.16 patients received enucleation as the primary treatment (operation group),25 eyes received chemotherapy before enucleation (chemotherapy group).There was no significant statistical difference between two groups for the gender,unilateral and bilateral,international staging or diagnostic age (P＞0.05).The histopathology report was performed to assess the risk of postoperative tumor-node-metastasis staging (pTNM) in each patient,and the extent of tumor invasion in the optic nerve,choroid and anterior chamber was divided into 3 levels of low risk,medium risk and high risk.Five deaths were all in the group E with chemotherapy before enucleation.Using R software survival analysis software package survfit function,the application of Kaplan-Meier estimation method,DSS of RB children was calculated from the time of diagnosis,up to the date of the death of patient.DSS differences between chemotherapy,operation group and eye removal time (more than 3 months,less than 3 months) in group E RB children were analyzed.Results The proportion of high risk pTNM stage in chemotherapy group was significantly lower than the operation group.But there was no significant difference between the two groups in the overall risk classification (x2 =3.130,P=0.077).For group D eyes,the overall risk classification in chemotherapy group was significantly lower than the operation group (x2 =5.870,P=0.015).There was no significant difference between the two groups in the overall risk of group E eyes (x2 =0.020,P=0.889).The DSS in chemotherapy group and operation group were 0.71 and 1.00,respectively;the difference was significant (x2 =3.700,P=0.05).The DSS in children whose enucleation delayed for more than 3 months and children whose enucleation performed within 3 months were 0.64 and 1.00,respectively;the difference was significant (x2 =4.800,P=0.028).Conclusion Systemic chemotherapy did not reduce the risk of tumor invasion and metastasis in patients with advanced RB.Instead,it will reduce the DSS in group E eyes of RB.

Objective To investigate the optimal condition for transforming alkanna tinctoria pigment into shikonin. Methods Transformation rate of shikonin served as index. Transformation temperature, time, ratio of 2% NaOH to alkanna tinctoria pigment (v/w) was optimized. Results With ratio of 2% NaOH to alkanna tinctoria pigment being 4.5 mL·mg-1, temperature 35℃ and the reaction time 4 h, the transformation rate reached the highest, and the average transformation rate was 64.86%. Conclusion This method is easy and simple, and suitable for industrialized production.

Objective To construct lentiviral vectors containing peptide P1-GFP fusion genes.Umbilical cord derived mesenchymal stem cells were infected with lentivirus carrying peptide P1 and GFP fusion genes.To inject the targeted umbilical cord derived mesenchymal stem cells into mice and to detect GFP expression in the spleen.Methods Umbilical cord derived mesenchymal stem cells were cultured with adhered tissues of umbilical cord smaller than 1 mm3 . Lentiviral vector containing P1-GFP fusion genes with engineering technology was constructed and infected the umbilical cord derive mesenchymal stem cells.Targeted umbilical cord derived mesenchymal stem cells were intravenously injected in the mouse tail vein and after 18 hours GFP expression was detected with immunohistochamical staining of the spleen tissues.Results Harvested umbilical cord derived mesenchymal stem cells grew well in culture medium. Green fluorescence on umbilical cord derived mesenchymal stem cells were observed under fluorescence microscope at 18 hours after infected with lentivirus.Green fluorescence intensity of umbilical cord derived mesenchymal stem cells was increasing over time and reached a peak at 72 hours.Umbilical cord derived mesenchymal stem cells highly expressed CD105 (90.0%)/CD44 (98%) and CD73 (85.0%)/CD90 (98.5%) molecules.GFP expression was detected in the spleen after intravenous injection of targeted umbilical cord derived mesenchymal stem cells in the mice 18 hours later.GFP expressing cells intimately contacted with lymphocytes.Conclusions Targeted umbilical cord derived mesenchymal stem cells contain P1-GFP fusion genes are constructed.Targeted umbilical cord derived mesenchymal stem cells can be targeted to mouse spleen and intimately contact with lymphocytes after intravenous injection.Our results lay the groundwork for further studies.

ObjectiveTo investigate the anti-tumorigenesis function of rhDCN on the leukemia K562 cells in vitro and analyze the possible mechanism.Methods Exponential phase of K562 cells were transfected with pcDNA3.1(+)-DCN,and PBS,liposome alone,and pcDNA3.1(+) vector were as control groups.Morphology change of K562 cells was detected by Wright stain,and cell proliferation activity was detected by MTT. Cell cycle and apoptosis of K562 cells were assessed by FCM. The expression of apoptosis-related protein,including bcl-xl,Mcl-1 and Bax were detected by Western blot.ResultsWright stain showed that typical apoptotic morphology of K562 cells were observed in DCN transfected group.There were no morphological changes of apoptosis in other groups. MTT method results showed that proliferation inhibition rate of the transfected cells [24 h (16.14±1.08) ％,48 h (14.07±1.01) ％,72 h (20.29±1.19) ％]was higher than that of the other control groups (P ＜ 0.05).FCM results showed that the apoptosis index (20.15±1.31) ％of the DCN transfected group was higher than that of the other groups (P ＜ 0.05),and most of cells arrested in the G0/G1 phase (51.15±0.57) ％ (P ＜ 0.05).Western blot results showed the expression levels of Bax were increased while bcl-xl and Mcl-1 were decreased in pcDNA3.1(+)-DCN/K562 group.ConclusionrhDCN can inhibit the growth of K562 cells and induce the apoptosis.The effect of DCN on bcl-xl,Mcl-1,Bax may play a role in its mechanism.

It is difficult to diagnose and treat local recurrence of the rectal cancer after operation. The early diagnosis is crucial for the recurrence of rectal cancer after operation. MSCT perfusion imaging is valuable in the diagnosis of recurrence of rectal cancer after operation. The application of MSCT perfusion imaging following rectal cancer operation was reviewed in this article.

Objective To study the clinical diagnostic value of transvaginal ultrasonic in early cervical carcinema and cervical neoplasis.Methotis The suspected patients with cervical carcinoma and cervical neoplasis were detected with transvaginal ultrasonography,liquid based cytology and cervical biopsy.The sonograms of transvaginal ultrasonic were retrospectively analyzed.Results In early cervical carcinoma and cervical neoplasis,the diagnostic sensitivity of transvaglnal ultrasonic was 90.9%and 83.3%;and the diagnostic specifity of transvaginal ultrasonic was 70.6% and 60.0%;the rate of missed diagnosis of transvaginal ultrasonic was 9.1% and 16.7%.Conclusion Transvaginal ultrasonic plays an important part in the clinical diagnosis of early cervical carcinoma.

Objective To further clarify the clinicopathological, immunohistochemical and electron microscopic features, and prognostic aspect of a rare esophageal carcinoma, i.e., basaloid squamous carcinoma (BSC).Methods The archival materials of 763 cases of esophageal malignancies (1977-1996) from the Dept. of Pathology, General Hospital of Nanjing Military Region, PLA, were reviewed and sixteen cases (2.1%) of BSC were detected. The clinical and pathological features of these cases were evaluated. Immunohistochemistry (S-P method), histochemical staining, and electron microscope were utilized to further characterize the neoplasm.Results There were 9 males and 7 females, with a mean age of 58 years. The tumors were classified as stage Ⅰ (n=1), stage ⅡA (n=6), stage ⅡB (n=2), stage Ⅲ (n=5), and stage Ⅳ (n=2) according to the criteria of the UICC TNM classification system of malignant tumors (1987). Most neoplasms in our series were located in the middle third of the esophagus. Grossly, they had an appearance similar to that of conventional esophageal carcinoma. Histologically, they showed a typical cytoarchitectural pattern of BSC. The most important histologic feature of this tumor was that the carcinoma had a basaloid pattern, intimately associated with squamous cell carcinoma, dysplasia, or focal squamous differentiation. The basaloid cells were round or oval in shape with scant cytoplasm. The nuclei showed pleomorphism, hyperchromatin, and numerous mitotic figures. The basaloid cells were arranged mainly in the form of solid, smooth-contoured lobules with peripheral palisading. Comedo necrosis in the islands of basaloid cells, small cystic spaces containing Alcian blue or PAS positive material, and PAS positive hyalinized stroma were frequently found. A panel of immunostains were used for the basaloid component of the tumor with the following results: cytokeratin (Pan),14/16 (+); epithelial membrane antigen, 16/16 (+); Vimentin, 4/16 (+); S-100 protein, 7/16 (+). Carcinoembryonic antigen and smooth muscle actin were negative. Electron microscopic (EM) study of 7 cases revealed that the basaloid cells are poorly differentiated, with a few desmosomes and fibrils and numerous free and polyribosome. The microcystic and intertrabecular spaces identified by light microscopy were seen under EM to be lined by basal membranes and filled with either loose reduplicated or compact globoid basal lamina showing fingerprint-like pattern. Of the 11 cases with adequate follow-up, 8 cases died within 2 years, the average survival time being 16.2 months. No cases of stages Ⅱ, Ⅲ, or Ⅳ survived beyond 5 years. The one-year survival rate was 60% and two-year 20%.Conclusion It is indicated that the BSC of the esophagus is a distinct clinicopathological entity with poor prognosis. The cellular differentiation and biologic behavior of the esophageal BSC were assumed to be situated between those of conventional squamous cell carcinoma and small cell carcinoma.