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Method for quickly separating liver cells in portal area and central vein area from liver

A central vein and hepatocyte technology, applied in the medical field, can solve the problem of inability to separate hepatocytes in the portal duct area from the liver, and achieve the effects of simple operation, high purity and high viable cell rate.

Inactive Publication Date: 2019-03-12
THE FIRST AFFILIATED HOSPITAL OF ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The method of the invention solves the problem that the hepatocytes in the portal area and the central venous area cannot be separated from the liver, and at the same time, the operation is simple, and the obtained primary hepatocytes have high purity

Method used

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  • Method for quickly separating liver cells in portal area and central vein area from liver
  • Method for quickly separating liver cells in portal area and central vein area from liver
  • Method for quickly separating liver cells in portal area and central vein area from liver

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1 Liver tissue processing

[0065] 1. Disinfect all consumables with 70% ethanol, including the inside of the constant flow pump;

[0066] 2. Inject pentobarbital sodium solution (0.5%, about 0.7ml for 50g mice) intraperitoneally to anesthetize the mice;

[0067] 3. Open the abdominal cavity, ligate the branches of the portal vein, ligate the subhepatic vena cava (live), and ligate the arteries if possible;

[0068] 4. Insert the catheter into the portal vein;

[0069] 5. Wash the intrahepatic blood with solution Krebs / Henseleit buffer at a rate of 35ml / min (210ml / h) at 37°C;

[0070] 6. Continuous perfusion for about 10 minutes, while inserting a second catheter into the inferior hepatic vena cava;

[0071] 7. Use a syringe to push Digitonin solution (1ml); (to separate portal area cells, the perfusion direction is reverse perfusion from the inferior vena cava; to separate central venous area cells, the direction is to perfuse forward from the portal vein...

Embodiment 2

[0087] Example 2 Purification of hepatocytes

[0088] 1. The cells obtained in Example 1 were prepared into a suspension with solution A containing Percoll cell separation fluid, put into a 50ml centrifuge tube, centrifuged at 50g for 3.5min, and removed the supernatant;

[0089] 2. Resuspend with solution A containing Percoll cell separation medium, and centrifuge again;

[0090] 3. Repeat step 2 twice;

[0091] 4. Collect cells and resuspend cells in basal medium;

[0092] 5.50g, centrifuge for 5min, repeat the centrifugal purification twice, the cells are stained with trypan blue, counted, according to 1x10 6 / 25cm 2 The number of cells in the bottle, using complete medium + 20% FBS in 5% CO 2 incubator at 37°C.

[0093] 6. Change the liquid after 18 hours, and change the liquid every day thereafter.

[0094] Trypan blue staining results figure 2 . Viable cell rate >95%.

Embodiment 3

[0095] Example 3 Marker gene expression

[0096] qRT-PCR detection of liver cell marker genes Glul and CYP1A2 in the central venous region (PV), and liver cell marker genes Arg1 and Pck1 in the portal region (PP) in two groups of isolated hepatocytes. See the test results image 3 .

[0097] The expression of hepatocyte marker genes Glul and CYP1A2 in the central venous area was much higher than that in the hepatocytes in the portal area. On the contrary, the expression of liver cell marker genes Arg1 and Pck1 in the portal area was much higher than that in the central venous area. This indicates that the cells we isolated are of high purity.

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Abstract

The invention belongs to the field of medicines, and specifically relates to a method for quickly separating liver cells in a portal area and a central vein area from liver. The method comprises the following steps: pouring a Digitonin solution from different directions and subsequently purifying cells to obtain target cells; when the cells in the portal area are separated, killing the cells in the central vein area by Digitonin without influencing the cells in the portal area, and subsequently performing separation and purification to obtain the cells in the portal area; and when the cells inthe central vein area are separated, killing the cells in the portal area by the Digitonin without influencing the cells in the central vein area, and subsequently performing separation to remove redblood cells and dead cells in blood vessels so as to obtain the cells in the central vein area. The method solves the problem that the liver cells in the portal area and the liver cells in the central vein area cannot be separated from liver, and is simple to operate, and the obtained primary liver cells are high in purity and high in survival rate.

Description

technical field [0001] The invention belongs to the field of medicine, and in particular relates to a method for rapidly separating hepatocytes in the hepatic portal area and the central vein area. Background technique [0002] The structure of the liver is more complex. The hepatic veins originate from the central veins of the hepatic lobule, converge step by step into the left, middle, and right hepatic veins, and flow into the inferior vena cava, and the small veins in the caudate lobe and its vicinity directly flow into the inferior vena cava. The portal vein enters the liver through the hepatic portal, branches repeatedly and finally flows into the hepatic sinusoids of the hepatic lobules. Therefore, the hepatic vein and the portal vein run in opposite directions. The closer the hepatic vein is to the diaphragm, the larger the caliber, and finally merge into the three main trunks to flow into the inferior vena cava; the closer the portal vein is, the larger the caliber...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/067C12N2509/00
Inventor 陈叶苗肖靖芳张玉君卞修武
Owner THE FIRST AFFILIATED HOSPITAL OF ARMY MEDICAL UNIV
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