CN101991610A - method for structuring hepatitis E infection model - Google Patents
method for structuring hepatitis E infection model Download PDFInfo
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- CN101991610A CN101991610A CN2009100904984A CN200910090498A CN101991610A CN 101991610 A CN101991610 A CN 101991610A CN 2009100904984 A CN2009100904984 A CN 2009100904984A CN 200910090498 A CN200910090498 A CN 200910090498A CN 101991610 A CN101991610 A CN 101991610A
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Abstract
The invention discloses a method for structuring a hepatitis E infection model, which comprises the following steps of: infecting Meriones unguiculatus with hepatitis E viruses to obtain the hepatitis E infected model. The infection method is oral administration. The method of the invention has low price, adopts the Meriones unguiculatus infection model which is convenient to feed and test, and avoids the defects of high price, limited sources, high experiment requirement condition, long propagation period and the like of non-human primates used at present. The invention provides the ideal infection model for the study of HEV (hepatitis E virus) pathogenesis, vaccine development, interspecies transmission mechanism and the like.
Description
Technical field
The present invention relates to a kind of construction method of hepatitis E infection model, particularly a kind of construction method of hepatitis E meriones unguiculatus infection model.
Background technology
So far, in the research of hepatitis E, also lack the cell line of successfully cultivating hepatitis E virus (HEV), HEV research mainly still relies on experimental animal.Application of model such as primate such as macaque, chimpanzee, make HEV research in the virus replication site, toxin expelling and viremia persistent period, antibody growth and decline rule, genotype, the pathogenic difference of hypotype and kind propagate aspect such as plane mechanism more and made significant headway.Yet, primate exist cost an arm and a leg, originate limited, requirement for experiment condition is high, be difficult to shortcoming such as use in enormous quantities.For a long time, the various countries scientist attempts setting up a kind of rodent infection model, but does not succeed always.
At present the non-human primate infection model that uses cost an arm and a leg, originate limited, requirement of experiment condition height, the breeding cycle is long, test operation is complicated, raising is inconvenient, makes its application be subjected to great restriction.
Summary of the invention
The construction method that the purpose of this invention is to provide a kind of hepatitis E infection model.
The construction method of hepatitis E infection model provided by the present invention is with hepatitis E virus counteracting toxic substances meriones unguiculatus, obtains infecting the model of hepatitis E.
Described counteracting toxic substances method is oral.
The dosage of described counteracting toxic substances is 10
5-10
8Virus copy (genome dosage, Genomic doses) is with 10
6The virus copy is best infective dose.
Described counteracting toxic substances virus is gene 4 type hepatitis E viruss.
The present invention sets up meriones unguiculatus experimental infection model with gene 4 type HEV strains, and from the angle of comparative pathology, and it is similar to confirm that HEV infects gerbil jird histopathology pathological changes and people, pig infects HEV, sets up the meriones unguiculatus model of HEV infection.It is simple and easy to do that the meriones unguiculatus infection model has test operation, and feeding and management is convenient.Especially cheap, one of one thousandth to four percentage that 1 meriones unguiculatus only is a primate expense.Therefore, as the HEV infection model, obtained result of study is adding system, comprehensive, reliable more, has more cogency with meriones unguiculatus.
Experiment showed, with method of the present invention to make up the hepatitis E infection model that infect the back and 1 week viremia can occur, HEV is about sustainable 4 weeks in blood, the feces; Continue more than 7 weeks in the liver; Also can detect HEV RNA in the small intestinal.The HEV immunohistochemistry detects can find that as seen hepatocyte in blocks in the hepatic tissue of infected group gerbil jird is the positive reaction of HEV SABC, also have tangible positive signal in the tissues such as small intestinal, kidney, adrenal gland, testis.Clinical observation test Mus shows no obvious abnormalities performance, and pathological study finds that liver has congestion, and hepatocyte generally sees the degeneration that has degree different, pathological changes such as the focal shape infiltration of lymphocyte between lobules of liver portal area or hepatocyte.Intestinal, kidney, cardiac muscle, testis etc. are organized and are also seen have tangible histopathology to change.
Method of the present invention is cheap, use to raise, test operation meriones unguiculatus infection model easily, deficiency such as avoided the non-human primate of present use to cost an arm and a leg, limited, the requirement of experiment condition height of originating, breeding cycle are long.For HEV pathogenesis, vaccine development, research such as kind of mechanism of transmission provides a kind of ideal infection model more.
Description of drawings
Fig. 1 infects for HEV that HEV RT-PCR detects electrophoresis result in the feces, blood serum sample of meriones unguiculatus
Fig. 2 is the photo of HEV antigen immune group positioning result in the histopathology of HEV counteracting toxic substances gerbil jird and liver, the intestinal
The specific embodiment
Below in conjunction with instantiation the present invention is elaborated.Experimental technique among the following embodiment if no special instructions, is conventional method.
Following percentage composition is the quality percentage composition if no special instructions.
The structure of embodiment 1, hepatitis E infection model and compliance test result thereof
1, the structure of hepatitis E infection model
(1) test meriones unguiculatus (available from the department of the Chinese Academy of Sciences of laboratory animal section of the Capital University of Medical Sciences): select SPF level or cleaning level for use, the 50-80g meriones unguiculatus.After antibody test, select the negative Mus of HEV IgG, select 28 and be equally divided into two groups, 14 every group, shielding system is raised separately.
(2) counteracting toxic substances: select HEV gene 4 type viruses (its genomic sequence is to be numbered the sequence shown in the FJ409465 among the GENBANK) copy number 10
5Above pig liver pathological material of disease, preparation liver suspension, every meriones unguiculatus gavages 10 through the oral cavity
5-10
8Individual virus copy (genome dosage) is with 10
6Individual virus copy is best infective dose, and in the present embodiment, HEV content is 10 in every milliliter of suspension
6During individual copy, gavage 1 milliliter of above-mentioned liver suspension.Matched group gavages 1 ml physiological saline.
2, the structure compliance test result of hepatitis E infection model
(1) sample collection: hepatitis E meriones unguiculatus infection model behind step 1 counteracting toxic substances and matched group gerbil jird fecal specimens are collected once every day; Blood sample is gathered weekly once, eye socket blood sampling, separation of serum; Feces and serum are standby in-70 ℃ of preservations.Each group is put to death 2 gerbil jirds weekly, and the pathological changes situation of cuing open inspection, observing each organ, tissue is gathered two parts of hearts, liver, spleen, lung, kidney, lymph node, small intestinal sample, and a-70 ℃ of frozen HEV RNA that are used for detect; Another part usefulness 2.5% glutaraldehyde-paraformaldehyde mixed stationary liquid is fixed for pathological study and SABC location.
(2) infect HEV distribution in affirmation and the histoorgan: in order to confirm to infect, measure the persistent period of viremia, and the distribution situation of understanding HEV in each histoorgan, adopt HEV special primer (seeing HEV Chao Shi PCR special primer sequence table), reverse transcription Chao Shi PCR method is carried out the RT-PCR detection of HEV RNA to serum, feces, liver,spleen,kidney, large intestine, the small intestinal of test group.Gerbil jird only detects liver matched group.Extract total RNA in the sample to be checked with the Trizol method of routine, reverse transcription is to carry out nest-type PRC as template behind the cDNA to detect.Set up feminine gender, positive control (positive control is a template with the cDNA of HEV positive material, and negative control is the reaction system that lacks template) bring false negative with the failure that prevents to increase and pollute due to false positive.
HEV Chao Shi PCR special primer sequence table:
First round PCR primer (template is by sample cDNA to be checked):
HE164F1:5 '-GCR GTG GTT TCT GGG GTG AC-3 ' (R=A or G)
HE164R1:5 '-CTG GGM YTG GTC DCG CCA AG-3 ' (Y=C or T)
Second takes turns PCR primer (template is a first round amplified production):
HE137F2:5 '-GYT GAT TCT CAG CCC TTC GC-3 ' (Y=C or T)
HE137R25 '-GMY TGG TCD CGC CAA GHG GA-3 ' (M=A or C; H=A or C or T; D=A or G or T)
Reverse transcription Chao Shi pcr amplification program is:
1. reverse transcription program: 42 ℃ of 30min, 99 ℃ of 5min, 4 ℃ of 5min.
2. first round PCR:94 ℃ 2min; [94 ℃ of 30s; 58 ℃ of 30S; 72 ℃ 30; 30cycles]; 72 ℃ of 7min.
3. second take turns PCR: the same first round.
Be 137bp if second takes turns the amplified production size, then the result is positive findings for detecting virus.
(3) pathological study: make paraffin section, HE dyeing back observation by light microscope.Microsection manufacture carries out (" pathological examination technology " that Shanghai science tech publishing house published in 1978) according to a conventional method.
(4) HEV SABC location:
1. the routine paraffin wax section dewaxes to water through dimethylbenzene, and (0.2M pH7.2) washes 3 times 5min/ time to use PBS then; Then 3%H is put in section
2O
2Incubated at room 10min, behind distilled water flushing, (0.2M, pH7.2) flushing is 3 times, 5min/ time to use PBS then.To cut into slices then with normal bSA working solution sealing, incubated at room 15min, serum deprivation inclines, add the anti-people HEV of rabbit antibody (available from Beijing Bo Aosen Bioisystech Co., Ltd), hatch 1.5h for 37 ℃, PBS (0.2M, pH7.2) flushing, 5min/ time, 3 times; Drip biotin labeling two anti-working solutions, hatch 30min for 37 ℃, PBS (0.2M, pH7.2) flushing is 5min/ time, 3 times, drip the strepto-avidin working solution (available from Beijing Bo Aosen Bioisystech Co., Ltd) of horseradish peroxidase-labeled, hatch 30min for 37 ℃, and PBS (0.2M, pH7.2) flushing is 5min/ time, 3 times, the DAB 5-10min that develops the color; Distilled water stops, and dyes 2min with the haematoxylin lining, dehydration, and mounting, microscopically is observed.
The result is as described below:
(1) RT-PCR detects
Allly can detect virus, 6 weeks of toxin expelling time remaining to the since second in the hepatitis E meriones unguiculatus infection model feces.Infect the back and can detect virus in the blood, 3 weeks of viremia time remaining on the 4th day.Can stably detect virus in the gerbil jird liver in one to seven week of infection, can detect for 3 weeks in the small intestinal.Matched group liver testing result all is negative.Partial results as shown in Figure 1, the 1-13 swimming lane is gerbil jird fecal specimens (1-10) and a liver sample (11-13) behind the counteracting toxic substances, all amplification obtains 137bp (arrow indication band); M:DNA marker DL2000; The negative contrast of NC.
(2) histopathology changes
The duration of test in 7 weeks behind the hepatitis E meriones unguiculatus infection model counteracting toxic substances, aspects and contrast Mus no significant difference such as the searching for food of clinical observation test Mus, body temperature, weightening finish.That regularly carries out cuts open in the inspection observation process, and the test group liver has the variation of enlargement slightly of color and luster deepening, volume more, and other organ is not observed and significantly observed pathological changes.Pathological study is found, the hepatic tissue clear in structure of matched group gerbil jird, and the hepatocyte marshalling, and the liver of infection gerbil jird can be observed pathological changes in various degree.There are slight inflammatory cell infiltration, bile duct proliferation in 2 weeks behind the counteracting toxic substances based on the portal area, swelling of liver cell, and the hepatic cords arrangement disorder, the part central vein has congestion; In 4～7 weeks behind the counteracting toxic substances, show as multiple, focal lymphocytic infiltration.Later stage, hepatocyte had granular degeneration, vacuolar degeneration even necrosis, and Kupffer increases.The obvious congestion of matter and visible focal hemorrhage between kidney, the renal tubular epithelial degeneration, the generation coagulation necrosis that has, the kidney balloon expandable has protein cast in the renal tubules, stop up tube chamber; The Distal convoluted tubule vacuolar degeneration, proximal convoluted tubule karyopyknosis or dissolving disappear.The small intestine lamina propria edema, inflammatory cell infiltration, mucosal epithelium comes off, pathological changes such as intestinal villus fracture.
(3) HEV SABC location
Detect VAA result in each histoorgan of hepatitis E meriones unguiculatus infection model with SABC, all have positive material to distribute in gerbil jird liver that different times infects and the small intestinal, be strong positive.Also can detect positive signal in large intestine, spleen, kidney, adrenal gland, the testis tissue.In liver, positive material sees endochylema, karyon, but mainly is distributed in liver cytoplasm, is the slurry diffuse type and distributes.Can be observed the double-core hepatocyte of HEV antigen positive, positive hepatocyte is often big than normal liver cell.Also visible positive particle in Kupffer, the portal area bile duct epithelial cell.Partial results as shown in Figure 2, among Fig. 2, A: hepatitis E meriones unguiculatus infection model hepatic tissue medium-sized lymphocyte lumps is soaked into, (haematoxylin dyeing cut into slices 40 power microscopes under observed result); B: hepatitis E meriones unguiculatus infection model kiernan's space blood vessel peripheral lymphoid cellular infiltration, (haematoxylin dyeing cut into slices 20 power microscopes under observed result); C: most liver cell nuclears are the positive reaction of HEV SABC in the hepatitis E meriones unguiculatus infection model hepatic tissue, (immunohistochemical staining cut into slices 40 power microscopes under observed result); D: tangible HEV positive reaction signal (immunohistochemical staining cut into slices 40 power microscopes under observed result) is also arranged in the hepatitis E meriones unguiculatus infection model small intestine.
Claims (5)
1. the construction method of a hepatitis E infection model is with hepatitis E virus counteracting toxic substances meriones unguiculatus, obtains infecting the model of hepatitis E.
2. method according to claim 1 is characterized in that: described counteracting toxic substances method is oral.
3. method according to claim 1 and 2 is characterized in that: the dosage of described counteracting toxic substances is 10
5-10
8The virus copy.
4. method according to claim 3 is characterized in that: the dosage of described counteracting toxic substances is 10
6The virus copy
5. according to any described method among the claim 1-4, it is characterized in that: described counteracting toxic substances virus is gene 4 type hepatitis E viruss.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102830236A (en) * | 2012-08-29 | 2012-12-19 | 中国计量学院 | Method of screening medicament for treating allergic bronchial asthma |
| CN105287645A (en) * | 2014-07-15 | 2016-02-03 | 中国农业大学 | HBV (hepatitis B virus) meriones unguiculatus infection model construction method |
| CN109769748A (en) * | 2019-02-21 | 2019-05-21 | 昆明理工大学 | The construction method of Hepatitis E virus chronicity mouse model |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20070118000A (en) * | 2006-06-09 | 2007-12-13 | 다이이치 고교 세이야쿠 가부시키가이샤 | Photoelectric conversion element |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102830236A (en) * | 2012-08-29 | 2012-12-19 | 中国计量学院 | Method of screening medicament for treating allergic bronchial asthma |
| CN105287645A (en) * | 2014-07-15 | 2016-02-03 | 中国农业大学 | HBV (hepatitis B virus) meriones unguiculatus infection model construction method |
| CN109769748A (en) * | 2019-02-21 | 2019-05-21 | 昆明理工大学 | The construction method of Hepatitis E virus chronicity mouse model |
| CN109769748B (en) * | 2019-02-21 | 2021-07-06 | 昆明理工大学 | Construction method of hepatitis E virus chronic mouse model |
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