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Method for treating chronic liver diseases by using hepatocyte nuclear factor-4alpha (HNF4alpha)

A technology of hepatocyte nuclear factor and chronic liver disease, applied in the fields of cell biology, medicine, and molecular biology, can solve problems such as unclear, no clear report on the treatment of chronic liver disease, unclear reversal of liver fibrosis, etc.

Inactive Publication Date: 2010-11-24
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
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Problems solved by technology

However, so far, it has not been clearly reported to screen the important transcription factors related to liver fibrosis EMT, and regulate the activity of these transcription factors to inhibit the process of EMT and enhance the function of liver cells, thereby treating chronic liver diseases.
[0006] In addition, although it is known that some genes are differentially expressed in the process of liver fibrosis, it is not yet clear whether hepatic fibrosis can be reversed by regulating the expression of transcription factors, let alone which transcription factors should be regulated to reverse liver fibrosis. liver fibrosis process
[0007] In summary, there is still a lack of effective means to prevent and treat liver fibrosis, chronic liver disease and liver cirrhosis in this field. Therefore, there is an urgent need in this field to develop specific transcription factors closely related to liver fibrosis and their regulatory genes so that they can be used as Targets for specific regulation, thereby effectively preventing and / or treating liver diseases such as liver fibrosis

Method used

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  • Method for treating chronic liver diseases by using hepatocyte nuclear factor-4alpha (HNF4alpha)
  • Method for treating chronic liver diseases by using hepatocyte nuclear factor-4alpha (HNF4alpha)
  • Method for treating chronic liver diseases by using hepatocyte nuclear factor-4alpha (HNF4alpha)

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Experimental program
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Effect test

Embodiment 1

[0092] Expression of HNF4α and EMT-related indicators in different rat models of liver fibrosis and different developmental stages of liver fibrosis by immunohistochemical method

[0093] 1. Preparation of animal model of liver fibrosis:

[0094] DMN injury liver fibrosis model: Male SD rats were randomly divided into 4 groups, 10 in each group. Common food feeding, free drinking water, day and night lighting alternately. Group 1 received intraperitoneal injection of normal saline as the negative control group; Groups 2 to 4 were the liver fibrosis model group, and received intraperitoneal injection of 1% DMN solution at a dose of 10 μg / kg, three times a week for a total of 5 weeks. A rat liver fibrosis model induced by DMN was prepared. Rats were sacrificed at different time points, and the same parts of liver tissue were fixed with neutral formaldehyde and prepared as paraffin sections for immunohistochemical determination; the remaining liver tissue was frozen in liquid n...

Embodiment 2

[0100] Construction of recombinant replication-defective adenovirus AdHNF4α expressing HNF4α

[0101] 1. Obtain the HNF4α 1425bp cDNA fragment: design and synthesize primers according to the human HNF4α cDNA sequence. Sense primer (BglII restriction site added at the 5' end): 5'-CCG AGA TCT AGA ATG CGACTC TCC AAA ACC-3' (SEQ ID NO: 1). Antisense primer (EcoR V restriction site added at the 5' end): 5'-CGC GAT ATC GGC TTG CTA GAT AAC TTC CTG CT-3' (SEQ ID NO: 2).

[0102] The HNF4α cDNA fragment was amplified by PCR, and the product was electrophoresed on 1% agarose gel to identify the size of the fragment, and the gel was tapped and recovered and placed in an Eppendorf tube, and the weight of the gel was weighed. Add NT solution 200ml / 100mg gel to the Eppendorf tube, 50°C for 5-10min until the gel melts, pass the liquid through the column, centrifuge at 13,000rpm for 1min, add 600μl NT3 buffer, and centrifuge at 13,000rpm for 2min. 30 μl of double-distilled water was passed ...

Embodiment 3

[0109] Inhibitory effect of up-regulation of HNF4α expression on the process of liver fibrosis in rats

[0110] 1. Grouping of Liver Fibrosis Animals

[0111] DMN group: male SD rats were randomly divided into 4 groups, 10 in each group. Common food feeding, free drinking water, day and night lighting alternately. Group 1 was given intraperitoneal injection of normal saline as the negative control group; Groups 2 to 4 were liver fibrosis model groups, which were given intraperitoneal injection of 1% DMN solution at a dose of 10 μg / kg, three times a week for a total of 5 weeks (w), preparation of DMN-induced rat liver fibrosis model. Among them, the second group was set as the model control group; the third group was set as the blank virus AdGFP control group; the fourth group was set as the AdHNF4α-introduced group; 10 rats in each model group were sacrificed at 2w and 4w respectively. On the 4th week of DMN injection, 4×10 9 pfu AdGFP and 4 × 10 9 pfu AdHNF4α, sacrificed...

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Abstract

The invention relates to a method for treating chronic liver diseases by using hepatocyte nuclear factor-4alpha (HNF4alpha), in particular to a method for treating chronic liver diseases such as human hepatic fibrosis, and the like by utilizing the HNF4alpha and application of HNF4alpha. Researches shows that the progress of epithelial-to-mesenchymal transition (EMT) can be suppressed through regulating and controlling the gene expressions of the HNF4alpha in hepatocytes and hepatic stellate cells (HSC); and more importantly, the in vivo NHF4alpha adenoviral vector injection shows that the expression increase of the HNF4alpha has blockage and reversion effects on the hepatocytes and the HSC EMT in a hepatic fibrosis evolution and the fibrosis evolution fibrosis evolution, thus the invention provides a new means for preventing and treating the chronic liver diseases such as the hepatic fibrosis.

Description

technical field [0001] The invention relates to the fields of molecular biology, cell biology and medicine. Specifically, the present invention relates to the method and application of using Hepatocyte Nuclear Factor 4α (Hepatocyte Nuclear Factor 4α, HNF4α) to induce chronic liver disease and liver cirrhosis. The invention also relates to a method for introducing HNF4α gene and protein into liver tissue and cells of human chronic liver disease and liver cirrhosis and a means for increasing the expression of HNF4α in these tissues and cells. Background technique [0002] Chronic liver disease and liver cirrhosis are common chronic diseases in clinical practice. They are difficult to treat, have poor clinical prognosis, occupy huge medical resources, and are major diseases that endanger national health. my country is a country severely affected by liver disease. At present, there are about 120 million carriers of hepatitis B virus surface antigen, and about 38 million people ...

Claims

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Application Information

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IPC IPC(8): A61K38/17A61K48/00A61P1/16
Inventor 谢渭芬林勇岳海燕尹川陈岳祥
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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