Influence of ganglioside combined with methylprednisolone sodium succinate on efficacy and neurological function in patients with acute myelitis.

BACKGROUND: Acute myelitis (AM) can lead to sudden sensory, motor and autonomic nervous dysfunction, which negatively affects their daily activities and quality of life, so it is necessary to explore optimization from a therapeutic perspective to curb the progression of the disease. AIM: To investigate the effect of ganglioside (GM) combined with methylprednisolone sodium succinate (MPSS) on the curative effect and neurological function of patients with AM. METHODS: First, we selected 108 AM patients visited between September 2019 and September 2022 and grouped them based on treatment modality, with 52 patients receiving gamma globulin (GG) + MPSS and 56 patients receiving GM + MPSS, assigned to the control group (Con) and observation group (Obs), respectively. The therapeutic effect, neurological function (sensory and motor function scores), adverse events (AEs), recovery (time to sphincter function recovery, time to limb muscle strength recovery above grade 2, and time to ambulation), inflammatory factors (IFs) [interleukin (IL)-6, C-reactive protein (CRP), and tumor necrosis factor (TNF)-α] and other data of the two groups were collected for evaluation and comparison. RESULTS: The Obs had: (1) A significantly higher response rate of treatment than the Con; (2) Higher scores of sensory and motor functions after treatment that were higher than the baseline (before treatment) and higher than the Con levels; (3) Lower incidence rates of skin rash, gastrointestinal discomfort, dyslipidemia, osteoporosis and other AEs; (4) Faster posttreatment recovery of sphincter function, limb muscle strength and ambulation; and (5) Markedly lower posttreatment IL-6, CRP and TNF-α levels than the baseline and the Con levels. CONCLUSION: From the above, it can be seen that GM + MPSS is highly effective in treating AM, with a favorable safety profile comparable to that of GG + MPSS. It can significantly improve patients' neurological function, speed up their recovery and inhibit serum IFs.

Disordered Expression of Tight Junction Proteins Is Involved in the Mo-induced Intestinal Microenvironment Dysbiosis in Sheep.

To evaluate the molybdenum (Mo)-induced changes of intestinal morphology and the relationship of intestinal tight junction (TJ) proteins expression and intestinal barrier function, a total of 20 healthy sheep were randomly divided into five groups of four: 0, 5, 10, 20, and 50 mg/kg BW/day Na2MoO4·2H2O were administrated in five groups named control group, Mo 5 group, Mo 10 group, Mo 20 group, and Mo 50 group, respectively. After 28 days of Mo treatment, the duodenum, the jejunum, and the ileum tissue were collected. The histopathology and the developmental parameters were evaluated by hematoxylin-eosin staining. The intestinal epithelial cell DNA damage was detected by TdT-mediated dUTP nick end labeling assay. The intestinal glycoprotein and the goblet cells were analyzed by Alcian Blue-Periodic Acid-Schiff (AB-PAS) staining and PAS staining, respectively. TJ proteins were determined by immunofluorescence technology. Results showed that excessive Mo significantly decreased the small intestinal villus height (VH), crypt depth (CD), VH/CD, and mucosal thickness (P < 0.05 or P < 0.01) while induced the damage of DNA in small intestinal epithelial cells. Moreover, excessive Mo injured intestinal barrier function by decreasing the percent of glycoprotein distribution area (P < 0.05) and the relative density of intestinal goblet cells (P < 0.05). Mo treatment induced decreased (P < 0.01) expression of Zonula Occludens-1, Occludin, and Claudin-1. In conclusion, excessive Mo interfered with the expression of TJ proteins, inhibited intestinal epithelial development, and further aggravated the intestinal barrier function damage, leading to disturbing the small intestinal microenvironment balance.

CT manifestations of the coronavirus disease 2019 of imported infection versus second-generation infection in patients outside the original district (Wuhan, China) of this disease: An observational study.

To explore the discrepancy in computed tomography (CT) manifestations of the coronavirus disease 2019 (COVID-19) in patients outside the original district (Wuhan, China) between cases with imported infection and second-generation infection, 22 patients with COVID-19 from 2 hospitals in Nanchong, China, 938 km away from the original district (Wuhan, China) of this disease were enrolled. All patients underwent initial and follow-up CT after admission during the treatment, and were divided into 2 groups. Group A and B were composed of 15 patients with a history of exposure to the original district (Wuhan, China) in short-term (i.e., imported infection), and 7 with a close contact with the patients with confirmed COVID-19 or with the healthy individuals from the original district (i.e., second-generation infection), respectively. Initial CT features including extent score and density score between groups were statistically compared. We found that all patients in group A and 3 of 7 patients in group B had abnormal CT findings while 4 of 7 patients in group B had not. Patients with abnormal CT findings were more frequent in group A than in group B (P < .05). On initial CT, pure ground glass opacity (GGO), and GGO with consolidation and/or other abnormalities were found in 20% (3/15) and 80% (12/15) patients in group A, respectively, while 1 (14.3%), 2 (28.6%), and 4 (57.1%) had pure GGOs, GGO with focal consolidation, and normal CT appearances in Group B, respectively. Patients with extent and density scores of ≥5 were more frequent in group A than in group B (all P-values < .01). Additionally, 3 of 4 (75%) patients with normal initial CT findings had focal pure GGO lesions on follow-up. In conclusion, COVID-19 in patients with a history of exposure to the original district can be severer than with the second-generation infection on CT.

Retraction: Activation of endoplasmic reticulum stress promotes autophagy and apoptosis and reverses chemoresistance of human small cell lung cancer cells by inhibiting the PI3K/AKT/mTOR signaling pathway.

[This retracts the article DOI: 10.18632/oncotarget.12718.].

Effect of molybdenum on reproductive function of male mice treated with busulfan.

Infertility is a serious public health problem worldwide. Molybdenum (Mo) plays an important role in maintaining normal metabolism. To explore the therapeutic efficacy of molybdenum (Mo) on male infertility, 90 mice were randomly divided into control, busulfan and busulfan + Mo groups. The male mice in the busulfan and busulfan + Mo groups were exposed to busulfan (20 mg/kg body weight) with a single intraperitoneal injection to establish the infertility model. The sterile mice were successfully obtained 30 days after busulfan exposure. Then, the male mice in the busulfan + Mo group were given drinking water containing 20 mg/L Mo continuously for 42 days. At 72 Day after treatment, 30 mice in the three groups were tested for various indices, and 60 mice were mated with females in spontaneous estrus. Mo significantly reversed the thinner seminiferous tubules and disappeared tubule and germ cells. Mo also normalized previously abnormal levels of testosterone, estradiol, luteinizing hormone, superoxide dismutase, lactate dehydrogenase, malondialdehyde. Furthermore, expression levels expression of Bax, Bcl-2, caspase-3 and caspase-9 returned to control levels; and finally, Mo-treated sterile mice obtained offspring with normal number and gender ratio. These results suggested that Mo at 20 mg/L had a significant therapeutic effect on reproductive dysfunction in sterile mice. Its mechanism could via repair of damaged testicular structures, regulation of abnormal reproductive hormone levels, decreased oxidative stress or and resistance to cell apoptosis. Mo may be a new candidate medicine for treatment of male infertility.

Activation of endoplasmic reticulum stress promotes autophagy and apoptosis and reverses chemoresistance of human small cell lung cancer cells by inhibiting the PI3K/AKT/mTOR signaling pathway.

OBJECTIVE: This study aims to investigate the effects of endoplasmic reticulum stress (ERS) on autophagy, apoptosis and chemoresistance of human small cell lung cancer (SCLC) cells via the PI3K/AKT/mTOR signaling pathway. RESULTS: The expressions of ERS-related proteins (PEAK, eIF2α and CHOP) up-regulated, autophagy-related proteins (LC3, LC3-II and Beclin1) and apoptosis-related proteins (Bax and procaspase-3) down-regulated in NCI-H446 and H69 cells after tunicamycin treatment for 24 h. Compared with the blank group, the tunicamycin, BEZ235 and tunicamycin + BEZ235 groups exhibited decreased expressions of p-PI3K, p-AKT and p-mTOR, and increased expressions of autophagy-related proteins (LC3, LC3-II and Beclin1) and apoptosis proteins (Bax and procaspase-3), and the most obvious changes were observed in the tunicamycin + BEZ235 group. MATERIALS AND METHODS: CCK-8 assay was applied to select the best cell line from five SCLC cell lines (NCI-H446, H69, H526, H146 and H209). Finally, NCI-H446 and H69 cells were selected for further experiments. NCI-H446/CDDP and H69/CDDP were selected and divided into the blank group, tunicamycin (an ESR inducer) group, BEZ235 (inhibitors of PI3K/AKT/mTOR pathway) group and tunicamycin + BEZ235 group. Cell apoptosis was detected by flow cytometry. Autophagy was observed by fluorescence microscopy and flow cytometry. Western blotting was used to detect the expressions of ERS-related proteins, autophagy-related proteins, apoptosis-related proteins and PI3K/AKT/mTOR pathway-related proteins. CONCLUSIONS: Our findings provide evidence that the activation of ERS could promote autophagy and apoptosis and reverse chemoresistance of human SCLC cells by inhibiting the PI3K/AKT/mTOR pathway.

Differentially expressed microRNAs and affected signaling pathways in placentae of transgenic cloned cattle.

Placental deficiencies are related to the developmental abnormalities of transgenic cattle produced by somatic cell nuclear transfer, but the concrete molecular mechanism is not very clear. Studies have shown that placental development can be regulated by microRNAs (miRNAs) in normal pregnancy. Thus, this study screened differentially expressed miRNAs by the next-generation sequencing technology to reveal the relationship between miRNAs expression and aberrant development of placentae produced by the transgenic-clone technology. Expressions of miRNAs and mRNAs in different placentae were compared, the placentae derived from one natural pregnancy counterpart (PNC), one natural pregnancy of a cloned offspring as a mother (PCM), and two transgenic (human beta-defensin-3) cloned pregnancy: one offspring was alive after birth (POL) and the other offspring was dead in 2 days after birth (POD). Further, signaling pathway analysis was conducted. The results indicated that 694 miRNAs were differentially expressed in four placental samples, such as miR-210, miR-155, miR-21, miR-128, miR-183, and miR-145. Signaling pathway analysis revealed that compared with PNC, significantly upregulated pathways in POL, POD, and PCM mainly included focal adhesion, extracellular matrix-receptor interaction, pathways in cancer, regulation of actin cytoskeleton, endosytosis, and adherens junction, and significantly downregulated pathways mainly included malaria, nucleotide binding oligomerization domain-like receptor signaling, cytokine-cytokine receptor interaction, Jak-STAT signaling pathway. In conclusion, this study confirmed alterations of the expression profile of miRNAs and signaling pathways in placentae from transgenic (hBD-3) cloned cattle (PTCC), which could lead to the morphologic and histologic deficiencies of PTCC. This information would be useful for the relative research in future.

Dual effects of molybdenum on mouse oocyte quality and ovarian oxidative stress.

A sub-acute toxicity test was performed to investigate the effects of molybdenum (Mo) on ovarian function. ICR adult female mice were exposed to Mo by free access to distilled water containing the Mo at 5, 10, 20, and 40 mg/L for 14 days. Compared to the control group, M II oocyte morphology, ovary index, and ovulation improved within the 5 mg/L Mo group, but were negatively affected by Mo at 40 mg/L. Morphologically abnormal ovarian mitochondria were observed at ≥ 20 mg/L. These alterations accompanied the changes in superoxide dismutase (SOD), glutathione peroxidise (GPx), and malondialdehyde (MDA) levels in ovaries. In conclusion, Mo affects oocyte quality possibly through regulating ovarian oxidative stress in a dose-dependent manner. It appears that Mo may improve ovarian function at a suitable concentration, which might be a candidate for the treatment of female infertility.

Effects of molybdenum on sperm quality and testis oxidative stress.

In order to investigate the effects of molybdenum (Mo) on sperm parameters and testicular oxidative stress, the ICR strain of adult mice were exposed to different doses of molybdenum for a sub-acute toxicity test. Compared to the control, our results showed that the sperm parameters, including the epididymis index, sperm motility, sperm count, and morphology, increased by a moderate dose of Mo (25 mg/L), but were negatively affected at high doses (≥ 100 mg/L). In addition, the changes of sperm parameters were accompanied with changes of the superoxide dismutase (SOD) activities, the glutathione peroxidase (GPx) activities, and the malondialdehyde (MDA) levels in testes. In conclusion, Mo affects the sperm quality through regulating the testicular oxidative stress in a complex manner.

The effect of molybdenum on the in vitro development of mouse preimplantation embryos.

The object of this study was to investigate the effect of molybdenum on the development of mouse preimplantation embryos cultured in vitro. Zygotes were flushed from one outbred mouse strain (Kunming), and then were cultured in potassium simplex optimized medium (KSOM) containing 0, 5, 10, 20, 40, 80, 120, and 160 µg/ml of molybdenum for 5 days until the mid-blastocyst stage. The addition of ≤ 20 µg/ml molybdenum did not affect the blastocyst and birth rates. Molybdenum at doses of 40 µg/ml and higher significantly decreased the cleavage, blastocyst and birth rates, the average cell number, and significantly increased the proportion of degenerative blastocysts. At 120 µg/ml molybdenum inhibited the blastocysts development to birth. At 160 µg/ml molybdenum caused overall developmental arrest (up to 16-cells) of embryos and their massive degeneration. In conclusion, molybdenum negatively affected the development of embryos in a dose-dependent manner. With lower doses (≤ 20 µg/ml), mouse embryos were not apparently damaged. With very high doses (≥ 40 µg/ml), embryo quality significantly decreased. This assessment of the effect of molybdenum on the preimplantation embryo is an initial survey of toxicological risk.

Blastocysts cloned from the Putian Black pig ear tissues frozen without cryoprotectant at -80 and -196 degrees Celsius for 3 yrs.

The Putian Black pig, as one of elite cultivars of endemic species in China, has been on the verge of extinction and urgently needs protection. Somatic cell nuclear transfer (SCNT) and noncryoprotected frozen tissue technology have successfully resurrected several mammalian species. Therefore, this study explored the primary feasibility of conserving this breed using a combination of both technologies. Skin tissues obtained from the ears of adult Putian Black boars were frozen without cryoprotectant at -20, -80, or -196 °C and stored for 3 yrs. Primary cell culture, passage and subculture were performed on frozen samples after being rapidly thawed at 39 °C and on fresh pig ear tissues (control). Cloned embryos were reconstructed using fibroblasts (from frozen and fresh tissues) with enucleated oocytes. Live cell lines were obtained from tissues frozen at -80 and at -196 °C and appeared to have normal proliferative activity after passage; furthermore, they directed cloned embryos to develop to the blastocyst stage after nuclear transfer. We concluded that the population of Putian Black pig might be increased in the future by transferring cloned blastocysts into synchronized recipient pigs.

Inhibition of hepatitis B Virus replication by hepatocyte nuclear factor 4-alpha specific short hairpin RNA.

BACKGROUND: Previous studies showed that hepatocyte nuclear factor 4α (HNF4α) may play a critical role in hepatitis B virus (HBV) replication. AIMS: This study aimed to investigate the effect of knocking down of HNF4α with RNA interference technique on HBV replication in a HBV replication mouse model. METHODS: Four HNF4α, specific short hairpin RNA (shRNA)-producing plasmids were constructed. HBV mRNA and DNA replication intermediates were analysed using Northern and Southern blot respectively. The expression of HNF4α and HBV core antigen (HBcAg) was detected using immunohistochemistry technique. RESULTS: One of the HNF4α shRNAs, HNF4α shRNA1, efficiently inhibited the expression of HNF4α in HepG2 cells and mice liver. HBV RNA transcripts and DNA replication intermediates in HNF4α shRNA1 group were decreased 67.3 and 76%, respectively, in HepG2 cells, and 68.1 and 70.6% in mice liver respectively. The expression level of HBcAg in the liver was also decreased with the inhibition of HNF4α expression. CONCLUSIONS: These results suggested that decreasing of HNF4α expression was associated with the reduced level of HBV replication in HepG2 cells and mice liver. These data indicated that HNF4α played a critical role in HBV replication in vivo, and HNF4α shRNA could inhibit HBV replication in vivo.

Functional Characterization of Interferon Regulation Element of Hepatitis B virus Genome In Vivo.

The roles of interferon regulatory element (IRE) in Hepatitis B virus (HBV) genome on inhibitory effect of interferon against HBV are controversial in vitro. This study aimed to determine the functional characterization of HBV-IRE sequence in vivo. Wild-type or IRE-mutant HBV replication-competent mice were firstly established, and mice were subquently treated with polyinosinic-polytidylin acid (polyI.C) or phosphate-buffered saline via intraperitoneal. Results showed that PolyI.C inhibited viral replication, and increased the level of 2',5'-oligoadenylate synthase mRNA transcripts, a marker of INF-α/ß induction. Between wild-type and IRE-mutant HBV replication-competent mice, the levels of HBV-RNA and HBV-DNA replication intermediates were similar. After PolyI.C treatment, the decreasing of HBV-RNA was similar between two groups, but HBV-DNA replication intermediates decreased significantly less in IRE-mutant than wild-type HBV replication-competent mice. These findings suggested that IRE mutant reduced the inhibitory effect of interferon on HBV replication, which played a role in antiviral effect of interferon against HBV.

Application of hepatitis B virus replication mouse model.

AIM: To evaluate the value of the hepatitis B virus (HBV) replication mouse model with regard to several aspects of the study of HBV biology. METHODS: To evaluate the HBV replication mouse model in detecting the efficacy of anti-HBV agents, the interferon inducer polyinosinic-polytidylin acid (polyIC) and nucleotide analogues adefovir and entecavir were administered to mice injected with wild type pHBV4.1, and the inhibiting effect of these agents on HBV DNA replication was evaluated. To identify the model's value in a replication ability study of HBV drug-resistant mutants and a HBx-minus mutant, telbivudine resistance mutants (rtM204I, ayw subtype), adefovir resistance mutants (rtA181V + rtN236T, ayw subtype) and HBx-minus mutants were injected respectively, and their corresponding HBV DNA replication intermediates in mouse liver were assessed. RESULTS: Compared with the wild type HBV replication mouse model without antiviral agent treatment, the HBV DNA replication intermediates of the polyIC-treated group were decreased 1-fold; while in the entecavir- and adefovir-treated groups, the levels of HBV DNA replication intermediates were inhibited 13.6-fold and 1.4-fold, respectively. For the mouse models injected with telbivudine resistance mutant, adefovir resistance mutant and HBx-minus mutant, HBV DNA replication intermediates could still be detected, but the levels of HBV DNA replication intermediates of these mutants decreased 4.5-fold, 5.6-fold and 2.9-fold respectively, compared with the mouse model with wild type HBV plasmid. CONCLUSION: The HBV replication mouse model we established was a useful and convenient tool to detect the efficacy of antiviral agents and to study the replication ability of HBV mutants in vivo.

Prevalence and risk factors of fatty liver disease in Chengdu, Southwest China.

BACKGROUND: Fatty liver disease (FLD) is increasingly recognized as one of the most common chronic liver diseases in China. This study aimed to investigate the prevalence and risk factors of FLD in Chengdu, Southwest China, and to provide a relevant basis for the prevention and intervention of FLD. METHODS: Altogether 9094 subjects (4721 men and 4373 women) of over 18 years old who had received a medical checkup in the West China Hospital of Sichuan University between January and December 2007 were evaluated for FLD. FLD was diagnosed by ultrasonography. Body mass index (BMI), height, body weight, blood pressure, fasting plasma glucose (FPG), triglycerides (TG), total cholesterol (TCh), alanine aminotransferase (ALT), hepatitis B surface antigen (HBsAg), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured using routine laboratory methods. RESULTS: The overall prevalence of FLD was 12.5%, which was more than 3-fold higher in males than in females (18.9% vs. 5.7%, X2=359.624, P<0.001). The prevalence increased with age in females and males of less than 50 years. The prevalence of alcoholic, suspected alcoholic, and non-alcoholic FLD was 2.6%, 3.6%, and 6.3%, respectively. Multiple logistic regression analyses showed that 10 factors (male sex, age, BMI, FPG, hypertension, TG, TCh, HDL-C, LDL-C, and ALT abnormalities) were closely related to FLD. In heavy drinkers, obesity increased the risk of FLD by 23.78-fold (95% CI, 10.22-55.33), but heavy drinking was only associated with a 2-fold (95% CI, 1.50-2.66) increased risk in obese subjects. CONCLUSIONS: The prevalence of FLD among a health-checkup population in Chengdu, Southwest China was lower than the published for other areas of China. FLD in Chengdu adults was found to be closely associated with sex, age, BMI, and other metabolic syndrome features.

Establishment of cell lines using a doxycycline-inducible gene expression system to regulate expression of hepatitis B virus X protein.

The hepatitis B virus (HBV) X gene plays an important role in HBV-associated pathogenesis, especially hepatocarcinogenesis. Establishment of a stable and regulable HBx expression system will allow study of the function of this gene. Here, we describe the development of a doxycycline-inducible recombinant plasmid (pBPSTR3-FlagX) with the full-length HBV X gene and all components of the tetracycline-on ("Tet-on") gene expression system. This vector exhibited dose-dependent doxycycline-dependent induction of the Flag-HBx protein in HepG2 and Hep3B cells. We also observed dose-dependent doxycycline transactivation of HBx in HepG2 cells. After transfecting HepG2 cells with the pBPSTR3-FlagX plasmid, we isolated five puromycin-resistant cell clones with stable HBx expression, two of which exhibited stable and tight control of HBx expression by doxycycline. This new system has great potential for functional studies of the HBV X gene.

[Morphology and function of platelets stored in modified platelet additive solution at low temperature].

The aim of study was to evaluate the function of modified platelet additive solution (PAS-IIIM) with trehalose as a substitute of plasma for the storage of platelet concentrates at low temperature (10 degrees C). Apheresis platelets from 6 donors were divided and added with different media (group A: 100% plasma; group B: 70% PAS-IIIM/30% plasma; group C: 100% plasma/trehalose). Groups A, B, C were stored at 10 degrees C, 22 degrees C and -85 degrees C separately. In addition, group D (platelet concentrates stored with 100% plasma at 4 degrees C) was set up as control group for scan electronmicroscopy. The samples of each platelets were collected on day 0, 1, 5, 7 and 9 after storage respectively, while samples of platelets stored at -85 degrees C (group C) were collected on day 20 after storage. CD62p, hypotonic shock response (HSR), platelet aggregation, lactic dehydrogenase (LDH) and morphology of platelets were evaluated. The results showed that the expressions of CD62p in groups A and B increased in a time-dependent manner, but HSR and platelet aggregations decreased. The expression of CD62p, LDH release, and platelet aggregation in group A were significant higher than that in group B (p < 0.05). HSR in group A was significant lower than that in group B (p < 0.05). LDH release was significant high in samples of group C and the expression of CD62p was lower than that in other two groups (p < 0.05). It is concluded that the protective effects of 70% PAS-IIIM/30% plasma (10 degrees C) and plasma platelets (22 degrees C) on morphology of platelets are similar, but better than those of plasma platelets (4 degrees C) and plasma/trehalose (-85 degrees C). In short, PAS-IIIM serves as a good substitute of plasma for platelet storage, and protects the chilled platelets.

[Gene transfection of endostatin synergizes with doxorubicin to suppress human hepatocellular carcinomas: experiment with mice].

OBJECTIVE: To investigate whether endostatin, a potent antiangiogenic agent, synergizes with doxorubicin to suppress human hepatocellular carcinoma (HCC). METHODS: An endostatin expression plasmid, Endo-cDNA3.1, was constructed and transfected into COS-1 cells. Human HCC cells of the line HepG2 and human umbilical vein endothelial cells of the line HUVEC were cultured and stimulated by the supernatant of the CoS-1 cells transfected with Endo-pcDNA3.1 and doxorubicin of different concentrations. MTT method was used to detect the proliferation of the cells. (How many) BALB/c mice were inoculated with HepG2 cells to establish HCC models, and then divided into 4 groups to undergo intratumoral injection of pcDNA3.1, End-pcDNA3.1, doxorubicin, or doxorubicin + Endo-pcDNA3.1. Other mice were used as untreated control group. Two weeks later 5 mice from each group were killed with the tumors taken out. Immunostaining was used to calculate the microvessel density and Western blotting was sued to detect the expression of endostatin, hypoxia-inducible factor (HIF) and vascular endothelial growth factor (VEGF). RESULTS: The proliferation of the HUVEC cells, but not that of the HepG2 cells, transfected with Endo-pcDNA3.1 + doxorubicin was inhibited. Doxorubicin dose-dependently inhibited the proliferation of both HUVEC and HepG2 cells. Endostatin was strongly expressed in the cells treated with Endo-pcDNA3.1 the tumor size of the Endo-pcDNA3.1 and doxorubicin groups were (1545 +/- 180) mm(3) and (953 +/- 250) mm(3) respectively, both significantly lower than that of the untreated and pcDNA3.1 groups [(2360 +/- 330) mm(3) and (2235 +/- 268) mm(3), respectively, all P < 0.01], and the tumor size of the Endo-pcDNA3.1 + doxorubicin group was (426 +/- 87) mm(3), significantly lower than any other groups (all P < 0.01). The number of microvessels of the Endo-pcDNA, doxorubicin, and doxorubicin + Endo-pcDNA3.1 groups were all significantly less than those of the pcDNA3.1 and untreated groups (P < 0.05 or P < 0.01). The expression of HIF-1alphawas downregulated in the Endo-pcDNA3.1 and Endo-pcDNA3.1 + doxorubicin groups, but not in the doxorubicin group. The VEGF expression was down-regulated in the Endo-pcDNA3.1, doxorubicin, and Endo-pcDNA3.1 + doxorubicin groups, especially in the latter. CONCLUSION: Endostatin gene therapy synergizes with doxorubicin to suppress HCC.

Establishment and primary application of a mouse model with hepatitis B virus replication.

AIM: To establish a rapid and convenient animal model with hepatitis B virus (HBV) replication. METHODS: A naked DNA solution of HBV-replication-competent plasmid was transferred to BALB/C mice via the tail vein, using a hydrodynamic in vivo transfection procedure. After injection, these mice were sacrificed on d 1, 3, 4, 5, 7 and 10. HBV DNA replication intermediates in the liver were analyzed by Southern blot hybridization. The expression of hepatitis B core antigen (HBcAg) and hepatitis B surface antigen (HBsAg) in the liver was checked by immunohistochemistry. Serum HBsAg and hepatitis B e antigen (HBeAg) was detected by enzyme-linked immunosorbent assay (ELISA). Inhibition of HBV replication was compared in HBV replication model mice treated intraperitoneally with polyinosinic-polytidylin acid (polyIC) or phosphate-buffered saline (PBS). RESULTS: After hydrodynamic in vivo transfection, HBV DNA replication intermediates in the mouse liver were detectable on d 1 and abundant on d 3 and 4, the levels were slightly decreased and remained relatively stable between d 5 and 7, and were almost undetectable on d 10. The expression patterns of HBcAg and HBsAg were similar to that of HBV replication intermediate DNA, except that they reached a peak on d 1 after injection. No obvious differences in HBV DNA replication intermediates were observed in the left, right and middle lobes of the liver. After treatment with polyIC, the level of HBV intermediate DNA in the liver was lower than that in the control mice injected with PBS. CONCLUSION: A rapid and convenient mouse model with a high level of HBV replication was developed and used to investigate the inhibitory effect of polyIC on HBV replication, which provides a useful tool for future functional studies of the HBV genome.

[Optimization of culture measure for bovine-bovine and goat-bovine cloned embryos in vitro].

UNLABELLED: This study is conducted to explore an effective culture method for supporting the embryo development. The cattle fetal ear fibroblasts and the goat fetal ear fibroblasts are transplanted into the enucleated cattle oocytes separately by oocyte intraplasmic nuclear injection method to construct bovine cloned embryos and goat-bovine cloned embryos. The embryos are first cultivated in modified charles rosenkrans 2 amino acid medium (mCR2aa) and modified synthetic oviduct fluid medium (mSOF) separately. Then BSA (8 mg/mL) or FBS (10%) can be added to mSOF according to the different culture period. The supplements and orders, added during the first three days and after three days are as follow: BSA and BSA, BSA and FBS, FBS and BSA, FBS and FBS. On the basis of the cleavage rate, 8/16-cell rate, blastocysts rate and total cell number of blastocysts, the best culture way can be screened out. RESULT: First, cleavage rate, 8/16-cell rate, blastocysts rate and total cell number of blastocysts, cultivated in mSOF solution are all higher than those cultivated in mCR2aa( P < 0.05). Second, the cleavage rate and 8/16-cell rate, adding BSA and FBS into mSOF, are in turn 79.8% +/- 7.1%, 49.7% +/- 3.5%, 21.5% +/- 1.8%, and 115.2 +/- 4.3 in bovine cloned embryo, and 40.1% +/- 6.3%, 29.2% +/- 2.0%, 13.4% +/- 2.1% and 100.1 +/- 3.0 in goat-bovine cloned embryo, which are significant higher than other culture groups (P < 0.05). CONCLUSION: The goat-bovine cloned embryo can be cultivated by the optimized culture measure of bovine cloned embryo. The best culture ways of bovine cloned embryo and goat-bovine cloned embryo are all to use mSOF supplemented BSA in the first three days and then use mSOF supplemented FBS in the next five days.