ABSTRACT
Objective: To observe the morphological features of liver injury induced by hydrodynamic injection, and to explore the procedure and mechanism of liver repair. Methods: Twenty-five female Balb/c mice were intravenously injected with large volume of saline solution (1.8 - 2.0 ml) in 3 s. The mice were divided into 30 min, 8 h, 1d, 3d, and 7 d groups according to the post-injection time. Each group contained 5 mice. Six non-injected mice were used as control group. The blood samples from angular vein of the mice were collected to detect the levels of alanine transaminase (ALT). The morphological features of liver tissue of the mice in various groups were observed with HE staining. The proliferating cell nuclear antigen (PCNA) protein expressions in liver tissue of the mice in various groups were detected by immunohistochemical saining. The expression levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), epidermal growth factor (EGF), hepatocyte growth factor (HGF), transforming growth factor-a (TGF-α), Cyclin D1, vascular endothelial growth factor (VEGF), Bax and Bcl-2 mRNA were determined by Real-time PCR. Results: Compared with control group, the ratio of liver weight/original body weight in 8 h group was significantly decreased (P0.05). Compared with control group, the serum ALT level in 1 d group was significantly elevated (P0.05). Compared with control group, the liver tissue of the mice in 30 min group showed hepatocyte swollen and small hemorrhagic area, and the number of hepatocytes per high power field was significantly declined (q=4.760, P0.05). The hemorrhagic and necrotic areas almost disappeared in 3 d group. The histological appearance of liver tissue of the mice in 7 d group was same as control group. Compared with control group, the PCNA indexes in hepatocytes in 8 h and 1 d groups were significantly increased (t=4.458, P0.05). Compared with control group, the PCNA index in cholangiocytes in 30 min group was increased significantly (t=3.985, P0.05). The mRNA expression levels of TNF-a, IL-6, EGF, and VEGF in liver tissue of the mice in 30 min group were significantly higher than those in 8 h group (q=4.952, P<0.05; q= 14.750, P<0.01; q=14.750, P<0.01; q= 13.551, P<0.01). The HGF mRNA expression level in liver tissue of the mice in 3 d group was significantly higher than those in 30 min group and 8 h group (q=5.031, P< 0.05; q=4.631, P<0.05). The TGF-a mRNA expression levels in liver tissue of the mice in 8 h group and 1 d group were higher than that in 30 min group (q=4.592, P<0.05; q=8.137, P<0.01). The Cyclin Dl mRNA expression levels in liver tissue of the mice in 8 h group and 1 d group were higher than that in 7 d group (q=4.736, q=5.213, P<0.05). The Bax/Bcl-2 ratio of the mice in 1 d group was higher than that in 30 min group (q= 5.731, P<0.01). Conclusion: Acute liver injury induced by hydrodynamic injection mainly shows the hepatocyte swelling and hemorrhagic necrosis. The injury could be repaired naturally in a week. The various cytokines and growth factors related with liver regeneration are involved in the repair procedure.
ABSTRACT
Objective:To observe the morphological features of liver injury induced by hydrodynamic injection, and to explore the procedure and mechanism of liver repair. Methods:Twenty-five female Balb/c mice were intravenously injected with large volume of saline solution(1.8-2.0 mL)in 3 s. The mice were divided into 30 min,8 h,1 d,3 d,and 7 d groups according to the post-injection time.Each group contained 5 mice.Six non-injected mice were used as control group.The blood samples from angular vein of the mice were collected to detect the levels of alanine transaminase(ALT).The morphological features of liver tissue of the mice in various groups were observed with HE staining.The proliferating cell nuclear antigen(PCNA)protein expressions in liver tissue of the mice in various groups were detected by immunohistochemical saining.The expression levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),epidermal growth factor(EGF),hepatocyte growth factor (HGF),transforming growth factor-α(TGF-α),Cyclin D1,vascular endothelial growth factor(VEGF),Bax and Bcl-2 mRNA were determined by Real-time PCR.Results:Compared with control group,the ratio of liver weight/original body weight in 8 h group was significantly decreased(P<0.05),but there was no significant difference in 7 d group(P>0.05).Compared with control group,the serum ALT level in 1 d group was significantly elevated (P<0.01),but there was no significant difference in 7 d group(P>0.05).Compared with control group,the liver tissue of the mice in 30 min group showed hepatocyte swollen and small hemorrhagic area,and the number of hepatocytes per high power field was significantly declined(q=4.760,P<0.05)under microscope.Compared with 30 min group,the number of swollen cells was decreased in 8 h group,the hemorrhagic and necrotic area enlarged, both of the hepatocyte number and binuclear hepatocyte number per high power field were significantly increased (q=7.310,P<0.01;q=7.200,P<0.01).Compared with 8 h group,the hemorrhagic and necrotic areas in 1 d group were reduced,both of the hepatocyte number and binuclear hepatocyte number per high power field were significantly decreased(q=4.966,P<0.05;q=6.596,P<0.01);there were no significant differences compared with control group(P>0.05).The hemorrhagic and necrotic areas almost disappeared in 3 d group.The histological appearance of liver tissue of the mice in 7 d group was same as control group.Compared with control group,the PCNA indexes in hepatocytes in 8 h and 1 d groups were significantly increased(t=4.458,P<0.01;t=15.557,P<0.01);there was no significant difference in 7 d group(P>0.05).Compared with control group, the PCNA index in cholangiocytes in 30 min group was increased significantly(t=3.985,P<0.01);there was no significant difference in 7 d group(P>0.05).The mRNA expression levels of TNF-α,IL-6,EGF,and VEGF in liver tissue of the mice in 30 min group were significantly higher than those in 8 h group(q=4.952,P<0.05;q=14.750,P<0.01;q=14.750,P<0.01;q=13.551,P<0.01).The HGF mRNA expression level in liver tissue of the mice in 3 d group was significantly higher than those in 30 min group and 8 h group(q=5.031,P<0.05;q=4.631,P<0.05).The TGF-αmRNA expression levels in liver tissue of the mice in 8 h group and 1 d group were higher than that in 30 min group(q=4.592,P<0.05;q=8.137,P<0.01).The Cyclin D1 mRNA expression levels in liver tissue of the mice in 8 h group and 1 d group were higher than that in 7 d group(q=4.736, q=5.213,P<0.05).The Bax/Bcl-2 ratio of the mice in 1 d group was higher than that in 30 min group(q=5.731,P<0.01).Conclusion:Acute liver injury induced by hydrodynamic injection mainly shows the hepatocyte swelling and hemorrhagic necrosis.The injury could be repaired naturally in a week.The various cytokines and growth factors related with liver regeneration are involved in the repair procedure.
ABSTRACT
Objective To stimulate the expansion of natural killer cells (NK) in vivo, to achieve high expression of Flt-3 ligand ( FL) in the mouse liver by hydrodynamic injection technology , to obtain high purity of NK for cell therapy and then to purify the spleen lymphocytes by MicroBeads sorting ( MACS ) technique.Methods C57BL/6 mice were repeatedly treated with hydrodynamic injection of FL expression plasmid .Then, surface markers of splenic NK were analyzed by flow cytometry and purified by MACS technique .Results Compared with mice treated with one hydrodynamic injection, the spleen of mice treated with two hydrodynamic injections showed significant variation, with the absolute num-ber of lymphocytes increased (6.02 ±1.15) times, the proportion of NK subsets increased (2.07 ±0.39) times, and the absolute number of NK subsets increased (12.49 ±2.39) times.Cell surface marker detection confirmed that NK activity and surface markers did not change significantly .NK with a purity of about (83.81 ±0.92)％ was obtained by the combination of two magnetic beads sorted with CD 5 ( Ly-1) MicroBeads Positive selection and EasySep TM Mouse NK Cell Isolation Kit negative selection .Conclusion NK can be expanded in the spleen of mice by hydrodynamic injection of FL plasmid without influence on the NK activity and surface markers.The combined use of CD5 (Ly-1) MicroBeads positive selection with EasySep TM Mouse NK Cell Isolation Kit negative selection can effectively remove T , B and other miscellaneous cells, thereby contributing to obtaining high-purity NK. This study provides good reference for the immunotherapy of tumor cells.
ABSTRACT
Objective To observe the activation of anti-viral innate immune response of type Ⅰinterferon and inhibition of hepatitis B virus (HBV)genome replication in mice by HBV-3p-siRNA. Methods HBV-3p-siRNA was designed by targeting specific sequence of HBV S/P mRNA and was generated by in vitro transcription.Negative control siRNA (NC-siRNA)and non-modified HBV-siRNA were used as control groups.Blood samples were collected from tail vein of mice and the model of HBV-infected mice were established by hydrodynamic injection.Forty mice were divided into 4 groups with 10 in each group.The model group was only injected with pGL3.0-HBV1 .2 copy plasmid.The negative control group received peritoneal injection of NC-siRNA.HBV-siRNA group received peritoneal injection of HBV-siRNA and HBV-3p-siRNA group received peritoneal injection of HBV-3p-siRNA.The interferon-β(IFN-β)and hepatitis B surface antigen (HBsAg)in serum were detected by enzyme linked immunosorbent assay (ELISA).The copies of HBV DNA were assessed by fluore scence quantitative polymerase chain reaction (PCR ).The statistical difference between groups was determined using One way-ANOVA analysis by LSD or Dunnett T3.Results Serum level of IFN-β was (12.37±5 .32)pg/mL in model group,(22.61 ±6.29 )pg/mL in negative control group,(26.40±5 .39)pg/mL in HBV-siRNA group and (68.37± 21 .00 ) pg/mL in HBV-3p-siRNA group.The secretions of IFN-β into serum were significantly enhanced by HBV-siRNA and HBV-3p-siRNA compared with model group (F =23.988 and 46.523,respectively,both P <0.01).Serum level of HBsAg was (2 864.86±907.11 )ng/mL in model group,(2 198.86±456.89 )ng/mL in negative control group,(1 049.71 ± 396.28 )ng/mL in HBV-siRNA group and (640.86±383.08)ng/mL in HBV-3p-siRNA group.The expressions of HBsAg were inhibited by HBV-3p-siRNA and HBV-siRNA compared with model group (F = 23.537 and 39.144, respectively;P =0.025 and 0.010,respectively).Serum level of HBV DNA was (2.54 ×104 ±1 .46 × 104 )copy/mL in model group,(2.22×104 ±2.62×103 )copy/mL in negative control group,(3.59×103 ±2.88×103 )copy/mL in HBV-siRNA group and (2.65 ×103 ±1 .46×103 )copy/mL in HBV-3p-siRNA group.Serum level of HBV DNA were inhibited by HBV-3p-siRNA and HBV-siRNA compared with model group (F =15 .013 and 16.741 ,respectively,both P <0.05 ).All of the indicated siRNA used in the experiments showed no apparent effects on the body mass index of the mice models.Conclusion HBV-3p-siRNA,which induces the production of IFN-β and inhibits HBV replication through gene silencing in vivo ,may be a powerful bifunctional antiviral molecule.
ABSTRACT
In antiviral therapy of hepatitis B virus (HBV) infection, drug resistance remains a huge obstacle to the long-term effectiveness of nucleoside/tide analogs (NAs). Primary resistance mutation (rtM204V) contributes to lamivudine (LAM)-resistance, and compensatory mutations (rtL180M and rtV173L) restore viral fitness and increase replication efficiency. The evaluation of new anti-viral agents against drug-resistant HBV is limited by the lack of available small-animal models. We established LAM-resistance HBV replication mice models based on clinical LAM-resistant HBV mutants. Double (rtM204V+rtL180M) or triple (rtM204V+rtL180M+rtV173L) lamivudine-resistant mutations were introduced into HBV expression vector, followed by hydrodynamic injection into tail vein of NOD/SCID mice. Viremia was detected on days 5, 9, 13 and 17 and liver HBV DNA was detected on day 17 after injection. The serum and liver HBV DNA levels in LAM-resistant model carrying triple mutations are the highest among the models. Two NAs, LAM and entecavir (ETV), were used to test the availability of the models. LAM and ETV inhibited viral replication on wild-type model. LAM was no longer effective on LAM-resistant models, but ETV retains a strong activity. Therefore, these models can be used to evaluate anti-viral agents against lamivudine-resistance, affording new opportunities to establish other drug-resistant HBV small-animal models.
ABSTRACT
Objective:To prepare and test tetrameric sH-2K~d-HBc complex for the further measurement of the specific CTL response.Methods:PE labled streptavidin with 4 biotinylated binding sites can bind to 4 biotinylated monomer to form the corresponding tetramer.Mice were immunized via different methods of genetic immunization by use of the construted pcDNA3-C plasmid to get the specific CTLs.Then our prepared tetramer was applied to stain the specific CTLs by the analysis of flow cytometry.Results:We applied our prepared tetramer to stain the cells from the experimental groups and control group.The results showed the tetramer was able to discriminate the frequencies of specific CTL induced by the three immunol methods(0.24%,0.26%,0.36% vs 0.07%,P≤0.05).This demonstrated that the prepared tetramer could bind its targets specifically and efficiently.The three immunol methods induced different levels of immune responses.Compared with the traditional muscle injection,gene gun induced weaker humoral immune response and stronger cellular immune response,and hydrodynamic injection induced the strongest humoral and cellular immune responses.Conclusion:Have successfully constructed the sH-2K~d-HBc tetramer.The techniques and methods can be used for preparation of tetramers of other types of MHCⅠ molecules.
ABSTRACT
Objective To investigate the inducible ability of plasmid DNA carrying a RU486 regulatory system.Methotis Plasmid pRS22 containing RU486 regulatory system,liver specific promoter and transgene IL-12 was injected into mice by hydrodynamic injection.RU486 was injected intraperitoneally into mice at difierent time points after plasmid administration.The IL-12 1evel in serum was tested by an ELISA kit.The distribution and inducible expression of pRS22 in mice were assayed by measuring DNA,RNA and protein levels by PCR,RT-PCR and immunohistochemical staining.Resuits To determine the duration of the activity of plasmid pRS22,mice injected with 10μg of pRS22 were treated repeatedly with 250μg/kg of RU486 per 7 days after hydrodynamic injection of plasmid.IL-12 expression in serum abruptly increased to peak was detected at 10 h after induction and declined to baseline on day 6.Though peak values of IL-12 decreased gradually after each induction,IL-12 in serum could be induced until 15 weeks after plasmid administration.A total of 5μg of pRS22 was injected into mice to detect the effect of different induction manners on the IL-12 expression.The mice were treated with RU486 per day or per 2 days iil 6 days,respectively.The induction per 2 days resulted in a wavelike pattern of serum IL-12 expression with peak lev-els on the day of induction alternating with lower values on the following day.In contrast,sustained levels of IL-12 could be achieved by administering RU486 per day.Plasmid DNA and GLp65 mRNA were detected in liver of mice with or without RU486 until at least day 28 after plasmid administration.However,IL-12 p35 mRNA was detected only in the liver of mice with RU486 induction.IL-12 immunohistochemical staining in liver demonstrated that IL-1 2 expressed predominantly in the hepatocytes near the surface of liver or between the central vein and portal area after induction with RU486.In contrast,no IL-12 expression was observed in the hepatocytes after induction with sesame oil.Conclusion Tight temporal and spatial control of transgene IL-12 expression could be achieved by RU486 regulatory system driven by liver specific promoter.
ABSTRACT
Objective To evaluate the inducibility of target gene expression induced by plasmid DNA carrying a RU486 regulatory system. Methods Plasmid pRS-LacZ encoding LacZ gene and plasmid pRS22 encoding murine interleukin-12(IL-12)gene were used to bansfect cells in vitro or be rapidly injected into the vein of mice. Then LacZ expression was assayed in cultured cells and tissues by X-gal staining.The IL-12 level was tested in the supernatants of cultured cells and in the serum with ELISA. ResultsIn the presence of RU486.30％ of the liver derived hepatic HepG2 cells were dyed blue while less than 1％of the HepG2 cells were blue colored in the absence of RU486. Less than 0.5％ of other non-hepatic cells were in blue with or without RU486. After transfection, the IL-12 level in the supernatants of HepG2 cells increased with increasing concentrations of RU486 treatment. After intravenous injection of plasmid pRS-LacZ,blue colored cells were observed only in liver tissue while induced by RU486. After intravenous injection of plasmid pRS22,the IL-12 expression level in serum was related with the dosage of RU486 or/and plasmid DNA. Conclusion Plasmid DNA containing RU486 regulatory system could open or close IL-12 gene expression by the addition or deletion of RU486. And the IL-12 expression level in serum could be regulated by altering the dose of plasmid DNA or/and RU486.