JPS6094084A - In-vitro infection of liver cell due to hbv virus - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for oral infection of hepatocytes in vitro by hepatitis B virus (Hbv).

The present invention also relates to infected hepatocytes and their uses, particularly 1-1b
Concerning the use of infected hepatocytes for the study of v viruses and for the production of viral antigens.

As is well known, the liver is the main target of the hepatitis B virus in vivo. The virus multiplies in the liver and causes cell lysis due to an immune response mediated by lymphocytes.

Furthermore, it is known that the presence of a viral genome integrated into the genome of hepatocytes is closely related to the progression of the tissue to a cancerous state.

In order to elucidate these phenomena, we investigated oral infection.
It is understood that it is important to create a research model.

Recent technology has made it possible to introduce l-1bv genomic DNA into cultured mammalian cells. However, this mode of infection is not indicative of natural processes, and it is particularly difficult to study virus replication (cL). Furthermore, in order to investigate the infectious process, it is particularly important to have access to normal adult hepatocytes rather than hepatocytes collected from diseased livers.

French Patent Application No. 8307148 of April 29, 1983 in the name of the Applicant describes a system in which the behavior of infected hepatocytes can be studied over a long period of time, ie about 6 weeks.

This type of system is obtained in particular by co-culturing healthy human hepatocytes and rat epithelial cells.

The inventors have carried out research in this direction and have succeeded in studying the behavior of systems other than symbiotic culture systems and improving infection methods for such systems.

It is therefore an object of the present invention to provide a method by which hepatocytes can be infected in vitro with hepatitis B virus.

The object of the invention further comprises these infected cells,
The object of the present invention is to provide a model or tool particularly suitable for the study of v-viruses and the production of viral antigens.

A feature of the method of the invention is that human or large mammalian hepatocytes of the type obtained by a process involving enzyme perfusion of defined zones of the liver using the vasculature, or cultures made from these isolated cells, The method is performed as described above and includes the step of contacting the hepatocytes with Hby virus before or after culturing.

'Hby virus' in the text and claims
A product refers to either the complete virus, its complete DNA or antigen-encoding sequences.

It was surprisingly found that viruses and Hbv particles invade cells. That is, the presence of viral DNA within a cell can be determined from the viral antigen that may be present within the cytoplasm of the cell.

According to an advantageous embodiment of the invention, the freshly isolated hepatocytes or their cultures or co-cultures are not previously chemically treated before the infection step.

In particular, treatments that denature cell membranes and make it easier for viruses to invade seem unnecessary.

Studies of this infection step have shown that it is advantageous to ensure close direct contact between the liver cells and the virus.

For this, use hepatocytes or hepatocyte cultures whose conditions are similar to those obtained after mild desiccation. Preferably, the hepatocytes are sequentially washed and centrifuged, and the supernatant is removed and covered. If it is a culture, dry it slightly.

According to a preferred embodiment of the invention, May 3, 1983
Hepatocytes, such as those obtained by the method described in US Pat. No. 4,997,391, or cultures of such hepatocytes under conventional conditions, are used.

To obtain hepatocytes, a process is used that involves enzyme perfusion of selective zones of the liver, especially the left hepatic lobe, which represents about 10% of the total liver.

To effect perfusion, the vascular system, particularly the portal vein system, is used and the arterial system is ligated. A perfusion enzyme solution containing enzymes such as collagenase, trypsin, dispase or the like is injected using a means such as a cannula. Irrigation flow is approximately 40 to 60 d/min and approximately 15 to 2
Hold for 5 minutes and continue.

By this procedure, in which a part of the liver is purified with enzymes, healthy human hepatocytes can be obtained at a high yield of 1 (Δ-da of i to 101 Il cells).

Hepatocytes isolated as described above are desiccated or similarly treated and then treated with virus or cultured under conventional conditions. This culture is also contacted with the virus after drying or similar treatment.

Surprisingly, the infection efficiency of l'bV on freshly isolated hepatocytes by the above process [,1,JQ-cultured lζ
It has been found that the infection efficiency obtained for the cells can be at least equal to, if not greater than, the infection efficiency obtained for the cells.

The period of time during which the cells are contacted with the virus is about 30 to 60 minutes, particularly about 45 minutes.

It is preferable to carry out the treatment at room temperature, preferably under uniform stirring.

It is best to use infectious serum to attack viruses.

In this case, whole serum may be used, or as a modification, serum with increased zone particle density may be used.

For example, hepatocytes obtained as a centrifuge are suspended in serum.

About 2 to 4 x 10 cells per about 1004 serum, especially about 3xi
oUse a melon.

For desired applications, particularly for the production of viral antigens and/or the investigation of the behavior of 0-1bytes in infected cells, freshly isolated Jll cells are infected and infected cells are treated. Infected cells can be cultured under conventional conditions or treated with Jt according to the method described in the aforementioned French patent application.
Live culture.

For other applications, especially for testing the infectivity of serum, infected III cells can be used directly.

Therefore, by means of the method of the invention it is possible to obtain various systems for detecting 1''bv in a given medium and investigating the biology of viruses. A gel is provided that is particularly suitable for studying viral invasion, viral replication, viral integration, and viral antigen production. !Provides gel for researching 111 cancers thought to be JJ.

Therefore, one of the objects of the present invention is that there is a new product that is infected before culture or after infection. and virus anti-1
There is the use of infected Jl cells in the production of yeast cells.

Other features and features of the present invention will become clear from the dozens of embodiments and drawings.

11 Takumi Kami Isolation Method of Hepatocytes Livers were dissociated from 8 kidney donors aged 20 to 25 within a few minutes after nephrectomy according to the following conditions.

The portal vein and left branch of the portal vein were quickly cleaned, and a cannula was inserted to perfuse only the left hepatic lobe.

The artery from the left hepatic lobe was ligated to prevent backflow of blood and to reduce loss of perfusion solution, and the perfusion solution was drained through the vena cava.

Perfusion was started immediately.

The liver was first washed with 1) l-17,6 for 20 minutes at 37° C. with a flow of 175 d/min l-1 epes buffer.

Next, @epes, pl-1z, er buffer L ta 5
A 0.05% collagenase solution containing mM (7) CaCjt was perfused for 20 min without recirculation at a flow rate of 50 min.

Approximately 21 l''epes buffer 1 and 11 l of enzyme solution were used. No enzyme was added to the solution.

A well-perfused part of the liver was selected and transferred to LH5L eibovitz medium containing 0.2% bovine albumin and gently shaken out (to disperse the cells).

The cell suspension was filtered through gauze and allowed to settle for 20 minutes.

The supernatant, which contained enough blood and sinusoidal debris to constitute cell debris, was discarded, and the pellet was washed three times by centrifugation at 50Q for 5 minutes.

Cell yields reached as high as IOX 10 cells, with 70-85% of the freshly isolated cells viable as determined using the trichrome exclusion test and phase-contrast microscopy with the J-cell balance test. Was.

To test the reproductive capacity of hepatocytes, 10 n/me porcine insulin and 0.2% bovine albumin and 10%
of fetal bovine serum and 5×10 M of)-thizonehemisuccine-1-
Using 12 medium, fibronectin
coated or uncoated 2s
3 single twin cells per polystyrene flask of cti
It was inoculated at a ratio of xlO'fllj.

Parenchymal cells formed a monolayer of granular epithelial cells and survived until day 8. Electron microscopy showed micro111+411i structural characteristics of in vivo hepatocytes.

Hepatocytes from a 25-year-old female former donor were isolated according to the procedure of Example 1 and cultured in Petri plates. 7 in a medium consisting of 1.5 IIdl l-1a1-1a containing 10% fetal bovine serum.
×105 cells were used.

After two days of culture, the hepatocytes were incubated for 30 or 60 minutes in the presence of 100 μl of human serum positive for Ac+Hbs and AgHbe. Collect the medium daily 1,'
7'1IiI;Lt-IJimC[nIA-At+
5triaII-125and AbotL-Hbe,
The production of A t3 t-1bs and A(IHbc) was measured by radioimmunology.The incubation time of hepatocytes in the presence of 1lbV was 30 or 60 minutes. -b if f tL
, similar results were obtained.

Figure 1 shows HIIS secretion curve (), Hbe secretion tllI
FA (・−・・・) and albumin secretion curve (−−
−−) are respectively sword size.

The secretion of 1-1bs and Hbe was measured daily in infected cultures 2 or 6 days after inoculation, and the 1]/N ratio (P: cpm
In the infection medium, N:C11111 control [1-ml] was shown. The average value of the control is 1-■) S and 160 cp
m, 11bo was 177 cpm. Infectivity aspect) 1]/N ratio of h (99.4.1 for Mato 1bs) 1 for bO
It was 0.9. j? Lubumin secretion per day in 1m of culture medium! Shown as n per hit.

As shown in FIG. 1, m Ag+-1b5 and AgHbe corresponding to lfi were secreted during the first 24ff5.

The maximum amount of AQl'bs produced was at 50 days after infection.
This was doubled in cultures infected 6 days after inoculation. This production is due to immunofluorome 1-lii (11111+1L
When measured with Ir10ep+1010111 (!try), it seemed to be related to albumin secretion. Agl-1
The secretion of bs and A(]+-1be was maintained throughout the culture. Their intracellular location was demonstrated by indirect immunofluorescence.

No cytotoxicity was observed in WA. Secretion of A17l-1bs and Agl-1be was not detected in uninfected cultures of hepatocytes.

Secretory hepatocytes were isolated according to Example 1, dried, and infected with l-1bV. The infected hepatocytes were then co-cultured with rat epithelial cells using the method disclosed in the aforementioned French patent application.

Antigen production is shown in Figure 2.

The curve (s-a) corresponds to Hbs secretion and the curve (L-J
) corresponds to l-1be secretion and curve-i) corresponds to albumin secretion.

As shown in FIG. 2, the liver cells were infected, the virus multiplied inside them, and antigens were produced after about 30 days.

[Brief explanation of the drawing]

Figure 1 shows primary 18 human hepatocytes after l-1bv infection.
Graph showing the production of l-1bs antigen, l-1be antigen, and albumin in the culture. There is a diagram showing the production of 11bS and 1'bO in the albumin. Continuation of IW Ritoben Hani Toima Mura Gen 1st page ■Int, C1,' Identification code Internal reference number // (C
12N 5100 012th 1:91) 0 Inventor Andre Guillusot France, @ Inventor Mitchel Brel France, S.4 0 Inventor Nadine Fist Cheese France, 350
00 Rennes, Avenue du Mer, 435,000
・Rennes, Rue de Plante Fist 8 Procedural Amendment 8 (Method) 1. Indication of matter A'1 1982 Patent Application No. 203593
No. 2, Title of the Invention In vitro infection of hepatocytes by l-1by virus 3, Relationship with amendment A 9 Name of patent applicant Lucierche Medical 6, number of inventions increased due to amendment 7, subject of amendment pending application, inventor's address, name column and applicant's representative column, drawings, power of attorney and document of delivery 8, Contents of the amendment (1) A proper application form stating the address and name of the inventor and the representative of the applicant shall be supplemented as shown in the attached sheet. ■Replenish official drawings as shown in the attached sheet. - (No change in content) (3) Supplement the power of attorney and its translation as shown in the attached document. (4) Add the transfer deed and the same translation as attached.

Claims (1)

[Scope of Claims] (1) A human or large mammal of the type obtained by a process involving enzyme perfusion of defined zones of the liver using the vasculature to infect hepatocytes with Hbv virus in vitro. hepatocytes or cultures prepared from these isolated cells are used, and the hepatocytes are contacted with 1 (bV virus) before or after culturing. ■ A method according to claim 1, characterized in that the freshly isolated hepatocytes or their cultures or symbiotic cultures are not previously chemically treated before the infection step.■ Mild. Claims 1 or 2, characterized in that hepatocytes or cultures having conditions similar to those obtained by drying are used to ensure close direct contact between hepatocytes and the virus. (4) The contact of the cells with the virus is maintained at room temperature and under constant agitation for about 30 to 60 minutes, in particular for about 45 minutes. Section to third
The method described in any of the paragraphs. ■ Viral challenge is carried out with infectious serum, possibly with an increased concentration of zonal particles;
2 to 4 cells per serum 1()Oρ 10°, especially about 3x
The method according to any one of claims 1 to 4, characterized in that hepatocytes are suspended in the serum at a ratio of 1:1 to 1:1. The method according to any one of claims 1 to 5, characterized in that after infection of freshly isolated hepatocytes, the hepatocytes are cultured or co-cultivated. (7) Infected hepatocytes constituting a model for testing infectious serum or a model for researching the biology of viruses, characterized in that they are hepatocytes that have been infected before or after culturing. (8) Application of infected hepatocytes according to claim 7 for antigen production of 1-1 h virus.