Inhibition of HBV Infection in Mouse Model by Dual Small
Interfering RNA Treatment

TANG Hong-bin1 , WU Kai-lang1 , JIANG Kun2 , PENG Ling3

( 1. Laboratory animal centre, State Key Laboratory of Virology, Wuhan University , Wuhan, Hubei 430071, PR China ;
2. Hubei academy of scientific and technical information , Wuhan, Hubei 430071, PR China;
3. Hubei centre of disease control and prevention , Wuhan, Hubei 430079, PR China )

【Abstract】 Objective To explore the therapeutic potential of RNAi on mutation in HBV genome resulting in resistance when Small interfering RNA (siRNA) is futher developed as an anti-viral drug, we express simultaneously two different 21-bp hairpin siRNA duplexes that specifically attack the HBs and HBx genes of HBV, respectively, in vivo. Method We copy the acute HBV mice model in the study, using hydrodynamics in the mouse tail. All the plasmid DNAs were injected into the tail vein of 8- to 12-weeks-old mice in a volume of Ringer’s solution equivalent to 11% of the body weight of the mouse. The total volume was delivered within 5~7s. The same technique is used to treat with interfering RNA in vivo. The antigens and DNA of HBV are determined with ELISA, Immunohistochemical staining and real-time RT-PCR. Results The animals could have been infected by the 10, 20, 30, 40 μg HBV plasmid DNA each body after 2, 4, 7days by hydrodynamic injection. However, the titer of HBsAg in serum decreased with time markedly. The serum and liver sample of the no therapy group had been obtained in the day 3, 7after mice were infected. The HBSX siRNA caused 50% reductions in the levels of HBeAg, respectively, compared to the HBS siRNA, and the HBS siRNA also caused 50% reduction to levels measured in sera from mice injected solely with the pHBV plasmid. The inhibitory effect of pSilencer-2.1-U6-HBSX siRNA was steady in day 1,2,3 after therapy. We assessed the effect of dual siRNA on HBV gene expression histologically by immunohistochemical staining for HBcAg in liver sections taken from mice treated in the kinetic experiment described above. HBcAg-positive cells were scarce (>0.1%) at the 3 and 7-day time point in the control. However, they decreased altogether from the liver of dual and single siRNA treated mice. The results by real time PCR revealed a stronger anti-replication effect of dual siRNA with reduction rate of 75.8% than single siRNA with 62.8%, compared with that of the position control in day 7. The inhibition effect was still observed at 3 days after injection with dual siRNA. Conclusion This dual siRNA system provides a more powerful tool to inhibiting HBV replication in vivo.
【Key words】HBV；Dual siRNA；Hydrodynamics；Mice model
[中国比较医学杂志 2009，19（9）：1-6]


Preliminary Study on the Enhance Effect of ECMS on
Immune Function of O-Asia FMD Vaccine

LIU Di-wen

（Laboratory Animal Center，Zhejiang University，Hangzhou 310058，China）

【Abstract】 In many papers it was reported that ECMS is added into vaccine to used as adjuvant. The aim of the paper is to verify the availability of ECMS to enhance immune function of O-Asia vaccine，and to choice the concentration of ECMS in use and the maintain time of producing antibody.
35 DHP guinea pigs were divided into 7 groups，number 1 ～ 4 groups contain 2500 μg、500 μg、100 μg and 20 μg ECMS adjuvant with 0.5 mL FMD vaccine respectively；number 5 contains 100 μg QA adjuvant with 0.5 mL vaccine；number 6 is 0.5 mL vaccine group；Number 7 is blank control group. The inject dosage for every guinea pig is 0.5 mL. The second immune injection was gone on after 3 weeks from the first immune by the same way. The serum antibody level of O type FMDV VP1 and Asia I type FMDV in 3、6、14 weeks after the second immunity were determined by ELISA methods. The degree of serum dilute is 1:800.
The result shows that the antibody level of a number of experimental groups in two vaccines at 3、6、14 weeks is higher than vaccine control group，but the best immune concentration of ECMS is 100μg. O type FMDV antibody level induced by ECMS is higher，but the maintain time is shorter. Asia I type FMDV antibody level is lower，but the maintain time is longer. It is indicated that ECMS is efficacious for increasing immune effect，but for different vaccine ECMS has different effect.
【Key words】 ECMS；O type FMDV VP1 antibody；Asia I type FMDV antibody；Immune reaction [中国比较医学杂志 2009，19（1）：5－7]

Intelligent Rotation Behavior Monitor for Experiment Animal
LIU Xin, WU You, WANG Yan, SHA Hong
（Institute of Biomedical Engineering, Chinese Academy of Medical Sciences & Peking Union Medical College，
Tianjin 300192, China）
【Abstract】Objective Parkinson's disease (PD), characterized by a progressive and selective degeneration of dopaminergic (DA) neurons in the substantia nigra pars compacta, is the second most common neurodegenerative disorder. It occurs in most cases after the age of 50, and the incidence increases with the aging process. The major clinical symptoms of PD are static tremor, bradykinesia, muscular stiffness and disturbances in balance, etc. The research into the pathology and treatment of PD has become a hot spot in neurology. And many researchers have been studying on it for many decades. Since the first report by Ungerstedt in 1968 that introduced the method in modeling animals rotation was published, 6-hydroxy-dopamine, which can lead to animals’ rotational behavior, has been widely used as a tool for anatomical and functional studies on central monoamine neurons. Researchers often reckon this model as an animal model of PD, as some characters of the model are similar to that of PD. Since the medicine works to accelerate the synthesis of dopamine in brain, there is no doubt that the research on the model will result in further understanding the mechanism of PD and effective screening of anti-PD medicines.
Many methods can be utilized to detect animal rotation. The first report in China about modeling animal rotation by using 6-hydroxy-dopamine is in 1984. The majority of the current apparatus are based on traditional analog circuit, the sensors of which are self-mad. This directly leads to the limitation of the accuracy. Furthermore, circuits of amplifier, filter and data collecting are needed to perfect the instrument. In addition, these apparatus generally use interface of RS232 to communicate with the PC, which is slow in speed and poor in accuracy just because of the limitation of the data transmission. And the electrical resource must be specially designed to generate power. With all the above defects taken into account, an intelligently multi-channel apparatus is contrived to monitor animal rotation, with the utilization of advanced modern computer and controlling technologies. Firstly, rotary encoder is adopted to receive regular digital signals. This doesn’t demand the use of those circuits. Accordingly, the hardware circuit is simplified and measure accuracy is efficaciously improved. It is considered to be the first innovation of my design. And the second brightness is the utilization of the USB chip of PL2303, which provides power supply via USB interface. Consequently, the volume of hardware circuit is apparently reduced, while the expansibility of the whole system is guaranteed. The last innovation, the most characteristic in my design refers to the specially contrived MCU program to forgo the interrupting signal caused by jitter. As a result, the measurement accuracy is obviously improved. Methods A combination of hardware and software is utilized in the design, which refers to the using of serial port controls, USB and rotary encoder, modern computer science, with the combination of traditionally experimental methods and the software designed in VC language. Results A mini animal rotation monitor has been developed, which can continuously monitor animal rotation and intelligently display the monitoring result. The principle of the design indicates that the theoretical value of the recorded pulse number is supposed to be 500 for a circle. And integer number of impulse makes sense. The average number of the pulse in each group equals to 499 in both positive and negative direction. And statistical analysis indicates the relative error is less than 0.2%, which means the system is quite precise in measurement. The more circles it rotates, the less of the relative error is. In other words, when the rotation circles increases, the detected impulse numbers get closer to the theoretical value. Therefore, it is safe to come to the conclusion that the system can work continuously and precisely, which matches the application requirement perfectly. Based on the above discussion, the rationality of the design is demonstrated. Conclusions Statistic analysis of experimental data proofs the rationality and effectiveness of the system. The rotational behavior monitor can play an importance role in research into the pathogenesis of PD and earching for anti-PD drugs. In this paper, based on a summary of extant systems monitoring animal behavior, an intelligent rotation monitor is designed to monitor animal rotation behavior. The system introduced can monitor animal rotation accurately and continuously. The main advantages of the system are small in volume, fast in signal transmission, easy in operation. Another outstanding superiority is that it can work continuously. And the following characters in design determine its advantages in operation. A rotary encoder works as the sensor. One of the two ends of the shafts is connected to the Velcro as the sensing part, which keeps away all disturbances from animal .The AT89C2051 MCU records data and communicates with PC via USB interface. The connecting chip Pl2303-USB not only qualifies for signal transmission, but also reduces the system volume effectively. As a result of, the connection to computer is simplified. With the assist of software programmed in VC language, the received data is stored in TXT file, which is convenient for backtracking and statistical analysis. The analysis of the uncertainty of experimental data shows the excellent performance of the system, which in return proofs the rationality of the design.[中国比较医学杂志，2009，19（3）：64-68]

The Anti-tumor Effect of New Kind of DHA Compound
and Its Effect on Caspase-3 Expression
LIU Yan1, YAO Wen-huan2, SUN Ke-ren3
(1. National Institute for the Control Pharmaceutical and Biological Products,
Beijing 100050,China; 2.Shandong Center for Disease and Prevention, Jinan
250014,China; 3. School of Public Health Shandong University, Jinan 250012 ,China)

【Abstract】Objective Malignant tumor is the kind of disease severely influencing health and quality of life of mankind. During the last twenty years, cancer death rate rise approximately 30%, while there is no ultimate breakthrough on the pathogenesis and therapy untill recently. At present, drug induced tumor cell apoptosis is one of the effective therapeutic measures. Here we report a new kind of DHA compound (AT-158) which used deepsea fish oil as key component, evaluate its antitumor effect and discuss the possible mechanism. Methods The animals selected for this study were 67 healthy Kunming mouses (weight 18 ～ 22 g), and hepatoma was established by subcutaneous injection of HepA22 cells.Then they were divided into six groups(Ⅰtumor control; Ⅱ~Ⅲ Cyclophosphamide(CTX)treatment groups; Ⅳ-Ⅵ AT-158 treatment groups). The size of tumors was monitored and they were harvested after the mice had been killed.The antitumor effect was assessed by the antitumor rate test, and then by using Western blot methods, the expression of Caspase-3 (a kind of apoptosis associated protein) in neoplasitc tissue was detected. We cultrued Hela(cervical carcinoma cell line) cell line in vitro and treated them with carboxymethyl cellulose(menstruum,CMC),CTX and AT-158(0.162 μg/mL, 0.323 μg/mL, 0.645 μg/mL, 1.29 μg/mL, 2.58 μg/mL, 4.30 μg/mL) respectively to assay the inhibitory effects on the cell growth by using MTT method[3]. And we went further to study the structural alterations on nucleus by fluorescence-microscope. Results There was significant difference between group Ⅰ and other five groups. The antitumor rate of group Ⅱ to Ⅵ was 69.15%,42.28%,56.05%,46.27%,31.28% respectively. The viability of Hela cell showed distinct inhibitation with the increase of concentration of AT-158, which appeared to be a good dose-effect relationship. The IC50 was 0.9814μg/mL（95%CI: 0.8754-1.0874 μg/mL）. Acridine orange was added to Hela cells. We noticed that eugonic tumor cell showed round nucleolus, while after treated with AT-158, the nucleolus seemed condensing and splitting, which was typically morphological changes during apoptosis. The levels of Caspase-3 expression was increased after AT-158 treatment, which was significant different with negative control group. The CTX treatment group also showed protein expression increase, but was not as good as AT-158 group. Discussion Cell can die in two ways: in a disorderly, destructive process called necrosis or by apoptosis, an orderly series of biochemical events that neatly eliminate the cells. Nowadays, with the deep research on the tumor molecule biology, it has been widely known that the occurrence of tumor is not only associated with the disturbance of proliferation and differentiation but also related to the turbulence of apoptosis. Caspases-3 happened to be one of the important protein involved in the regulation of apoptosis.Many laboratory studies suggest that n-3 fatty acids, especially the long-chain polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have antitumor effects. The mechanisms may involve the suppression of the biosynthesis of proinflammatory molecules, the influence on transcription factor activity and gene expression, the influence on signal transduction, the alteration of hormone-stimulated cell growth and the suppression of the production of free radicals and reactive oxygen species. AT-158 is a kind of anticarcinogen with DHA as the main ingredient.It was concluded that AT-158 could effectively inhibit the growth of some kind of tumors, which could partially be explained by its induction of programmed cell death. This study indicate that AT-158 is a potent and promising anticarcinogen with a broad spectrum.[中国比较医学杂志，2009，19（3）：25-26]

Research on Urinary Calculus Models on Rats induced by the Melamine

ZOU Yi-hai1,ZHANG Wei2,XU Zhi-wei1,FU Lu-di1,ZHANG Zhong-guang1,
HUANG Wu-dang1,HE Guo-zheng1,CHEN Jia1, WANG Xiao1, AO Hai-qing1

(1.Guangzhou University of TCM,Guangzhou 510405，China;2.Laboratory Animal Center,SUN YAT-SEN University, Guangzhou 510080,China)

[Abstract] Objective To observe the effect of urinary calculus induced by the Melamine fed on rats per day from 1 day to 30 days. Methods 130 SPF SD rats were male and female in half, and their mean body weight is（200 24）g, supplied by the Laboratory Animal Center of Guangzhou University of Traditional Chinese Medicine (TCM). Their qualified number is 2008A002 issued by Guangdong Experimental Animal Supervision Institute. All rats were randomized into 5 Melamine groups with 20 rats per group，the solvent control group with 10 rats and the blank control group with 20 rats. Melamine is manufactured by Henan Junhua Development Co. Ltd., and its purity is 88% detected by Food laboratory of Guangdong Bureau for Exit-entry Inspection and Quarantine (qualified No. 4400/0805004). The rats of the 5 Melamine groups were fed the Melamine with the dosage range of 0.05g/kg/d，0.1g/kg/d，0.2g/kg/d，0.3g/kg/d and 0.4g/kg/d，respetivily.The rats of the solvent control group were given 2mL of 10g/L methylcellulose distilled water by gavage per day.And the rats of the blank control group were given 2mL of distilled water per day.The experimental was carried out for 30 days. The experiment was carried out in a shielding facility（qualified No. 2008C063, issued by Guangdong Experimental Animal Supervision Institute） for animal experiment in Experimental Animal center of Guangzhou University of TCM. After continuous administration of melamine for 20 days, 10 rats were taken out from the each medication groups and the blank control group and the solvent control group. After 30 days, 10 rats were taken out from the each medication groups and the blank control group.The chosen rats were executed by incision of cervical vessels to induce bloodletting under ether anesthesia.The morphological changes of kidneys，ureters and bladders on rats were observed by naked eyes and stereomicroscope(Made by Olympas , Japan; Item No. SZ61).The body weight and organs index of each group as well as the level of serum CREA、BUN、UA、Ca、P、Mg were compared. The SPSS10.0 software processed all of the data. Results During the experiment all 130 rats were suvived. The manifestations of the rats which in medication groups after 12 days were as follows: loose hair, less activities, polyuria. And in some of the rats there were found that bloody urine, red and edema in the skin of posterior limbs, claudication. The levels of CREA in the medication groups(0.4g/kg/d after medication of melamine for 20 days,0.2g、0.4g/kg/d after medication for 30 days) were higher than those in the blank control group（P＜0.05）. The levels of BUN、UA in the 0.4g/kg/d group after medication for 30 days were higher than those in the blank control group（P＜0.05）.The levels of Ca、P in 5 medication groups after medication for 20 days were lower than those in the blank control group（P＜0.05）. The levels of Mg in 5 medication groups after medication for 20 days were lower,and the levels of Mg in 0.2、0.3、0.4g/kg/d medication groups were lower than those in the blank control group, the difference was significant（P＜0.05）.The rezults means that the levels of CREA BUN、UA、Ca、P、Mg were lower or higher lightly in the 0.4g/kg/d group than the blank control group. The weight of kidney on rats in 0.05、0.2、0.3、0.4g/kg/d groups after medication for 30 days were higher than those in the blank control group.The shape and colour of kedney in 5 medication groups were normal compared of the blank control group.The urinary calculus and yellow precipitates were not found in kidneys and ureters. But the punctiform and patchy hemorrhagic focus were odserved in some kidneys between cortex and medulla. Some congestion in the mucosa of bladder was found. The bladder calculus induced by the melamine were found in some male rats and the appearance rate was 52%（13/25）on 20th days as well as 56%（14/25）on 30 th days in 5 Melamine groups. The colour of the bladder calculus was yellow-white or white. Conclusions The male rats were fed the melamine with the dosage range of (0.05~0.4)g/kg/d for 30 days.Some male rats appearance the bladder calculus and the damage in kedneys were slight.
[Key words] Melamine；Disease Models；Urinary calculu；Rat
[中国比较医学杂志，2009，19（7）：5-10]

mir-34a may play a role in Alzheimer’s disease by affecting the expression of bcl2
WANG Xiao-ying, LIU Peng, ZHU Hua, XU Yan-feng,
MA Chun-mei, DAI Xiao-wei, LIU Ying, QIN Chuan
(Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences & Comparative Medical Center, Peking Union Medical College, Beijing 100021,China)

[Background] Alzheimer’s disease (AD) is a kind of neurodegenerative disease which is characterized by deposition of senile plaque, neurofibrillary tangles inside neurons and loss of neurons in hippocampus and cerebral cortex. About 50%–60% dementia cases are caused by this disease . It is reported that there are 2-4 million of AD patients in America and 17-25 million of AD patients all over the world. In the western countries, AD is the fourth cause that leads to death following the heart diseases, tumors and strok.The mechanisms of AD is not clear. The studies in present showed that APP, Tau, PS and ApoE may play an important role in the pathogenesis of AD. However, to our regret ,the treatments target APP, Tau, PS and ApoE don’t acquire the exciting effects. So it is necessary for us to understand the pathogenesis of AD from a new field.
MicroRNAs (miRNAs) are endogenously expressed small ssRNA sequences of about 22 nucleotides in length which naturally direct gene silencing by suppressing the translation and/or promoting the degradation of target messenger RNAs (mRNAs) by binding to their 3’-untranslated regions(3’-UTR) . microRNAs are abundant in the brain and play an important role in neurodevelopment and synaptic plasticity.The results of some experiments imply that the alteration of microRNAs expression may be involved in the pathogenesis of AD. However, the mechanism by which miRNAs affect the occurrence and development of AD is unclear.
mir-34a belongs to mir-34a family and this family consists of three highly related miRNAs expressed from two separate loci: miR-34a from chromosome 1p36 and the miR-34b/miR-34c cluster from chromosome 11q23. Various studies have shown that it is a potential tumor suppressor by inducing a G1 cell cycle arrest, senescence and apoptosis . Furthermore, miR-34a is located on 1p36, a region of frequent hemizygous deletion in human neuroblastomas and a variety of other cancers. However, the relationship between mir-34a and AD is not shown anywhere.
Bcl2 is an anti-apoptotic protein primarily expressed in mitochondria and the outer nuclear membrane, which prevents caspase-9 activation through an interaction with Apaf-1. It is neuroprotective against apoptotic cell death caused by amyloidogenic peptides . Overexpression of bcl2 could attenuate the processing of APP and tau and reduced the number of NFTs and extracellular deposits of Aβ . However, bcl2 gene expression regulation is not completely understood. Here, we provide data indicating that miR-34a contributes to bcl2 posttranscriptional regulation and that this pathway may be involved in the pathogenesis of AD.
[Methods] A miRNAs array was used for the analysis of microRNA expression from brain tissue of 3-month-old and 6-month-old APPswe/PSΔE9 mice. The results of microRNA array were validated by real time RT-PCR. The protein level of bcl2 in brain tissue from APPswe/PSΔE9 mice and control mice was detected by western blot. Through the construction of stable transfecant cell line of mir-34a and mir-34a knockdown, we showed that mir-34a could regulate the expression of bcl2. Through the experiment of bcl2 3’UTR-luciferase reporter, we showed the interaction of miR-34a with the 3’UTR of the bcl2 mRNA.
[Results] In our study we used the APPswe/PSΔE9 mice as a model for AD. miRNAs microarray was used for the analysis of miRNAs expression from brain tissue of 3-month-old and 6-month-old APPswe/PSΔE9 mice. The result shows that mir-34a was differentially expressed in transgenic mice versus control mice. Considering a false discovery rate of microRNA microarray, the expression level of mir-34a was also determined by real time RT-PCR in our lab. We used six APPswe/PSΔE9 mice and six age-matched controls and all reactions were run in triplicate. The result shows that mir-34a expressed at higher levels in 3-month-old and 6-month-old APPswe/PSΔE9 mice compared with age-matched controls.
We found by bioinformatic analysis and search of the TargetScan4.0 database (www.targetscan.org) that the bcl2 3’-UTR contains one putative binding site for miR-34a. To learn whether miR-34a can affect endogenous bcl2 protein levels, we first performed a Western analysis on the total protein extracts from the brain of 3-month-old and 6-month old APPswe/PSΔE9 mice. The results showed that the level of bcl2 protein was decreased both in 3-month-old and 6-month-old APPswe/PSΔE9 mice compared with age-matched controls, which is inversely linked to the expression of mir-34a.We also detected the mRNA level of bcl2 using real time RT-PCR and found that there are no difference in bcl2 mRNA level between APPswe/PSΔE9 mice and age-matched controls. This gave us a first hint that the over-expression of mir-34a might be the mechanisms acting to negatively regulate bcl2 in APPswe/PSΔE9 mice.
To validate the relationship between mir-34a and bcl2, we constructed the stable transfection cell lines that can constitutively express mir-34a using SH-SY5Y cells. Because the vector we used also contains an EmGFP (Emerald Green Fluorescent Protein) expression cassette, it is convenient for us to select cells expressing mir-34a through co-cistronic expression of EmGFP. The results showed that the stable transfection cell line of mir-34a clearly expressed high levels of mir-34a compared with controls. A Western blot performed on the same cells showed that bcl2 protein was remarkably reduced in the stable transfection cell line of mir-34a, as compared to cells transfected with the control vector. To study the relationship between mir-34a and bcl2 further, we knocked down mir-34a by transfecting SH-SY5Y cells with LNA antisense oligonucleotides targeting mir-34a and we analyzed the effects on bcl2 production. SH-SY5Y cells transfected with anti -34a LNA showed a significantly decrease in mir-34a level when compared to control cells transfected with LNA against a microRNA not expressed in these cells. As expected, the reduction of mir-34a was accompanied by an increase of bcl2 protein. We also detected the mRNA level of bcl2 in stable transfection cell line of mir-34a and SH-SY5Y cells transfected with anti-34a LNA, the results showed that the level of bcl2 mRNA was not affected by mir-34 overexpression or knockdown. This indicated that mir-34a could affect the expression of bcl2 protein by inhibiting the translation of bcl2 mRNA.
In order to test whether miR-34a can directly target bcl2 gene, we cloned the wild type 3’-UTR of the bcl2 gene downstream of the luciferase open reading frame. we used this luciferase-3’UTR reporter construct to cotransfect 293T cells with mir-34a expression vector or negative control vector. The luciferase activity assays at 48 hours post transfection showed that the over-expression of mir-34a strongly affected luciferase expression, measured as relative luciferase activity. However, when we used, as a reporter construct, a plasmid containing the 3’UTR of bcl2 mRNA where the core binding site for mir-34a was inactivated by site-directed mutagenesis, we observed only a very slight effect on luciferase activity. The result supports the bioinformatic prediction indicating the 3’UTR of bcl2 mRNA as a target for mir-34a.
[Conclusion] Our research is the first to show that mir-34a could regulate the expression of bcl2 through inhibiting the translation of bcl2 mRNA. Our results also suggested the abnormal expression of mir-34a may contribute to the pathogenesis of AD, at least in part by affecting the expression of bcl2. This provided us a new insight to understand the pathogenesis of Alzheimer’s disease.
Keywords: microRNA, mir-34a, Alzheimer’s disease, bcl2, APPswe/PSΔE9 mice
[中国比较医学杂志，2009，19（10）：5-10]