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Use of human recombination hepatic stimulator substance for reinforcing anti-apoptosis of liver cancer cell and application thereof

A technology of liver-stimulating factor and human recombination, which is used in medical preparations containing active ingredients, anti-tumor drugs, drug combinations, etc.

Inactive Publication Date: 2011-04-27
CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

HSS gene is expressed in many tissues such as liver, kidney, etc., but it can only specifically promote DNA synthesis in hepatocytes or liver-derived tumor cell lines, making it synthesized by G 0 phase enters the S phase, but has no stimulating effect on the proliferation of non-hepatic normal tissue cells and malignant cells in vivo or in vitro (Tang Hanman, He Fuchu: Research progress of cell-derived hepatocyte growth factor

Method used

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  • Use of human recombination hepatic stimulator substance for reinforcing anti-apoptosis of liver cancer cell and application thereof
  • Use of human recombination hepatic stimulator substance for reinforcing anti-apoptosis of liver cancer cell and application thereof
  • Use of human recombination hepatic stimulator substance for reinforcing anti-apoptosis of liver cancer cell and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Construction of vectors expressing human recombinant liver-stimulating factor

[0028] Using the mouse HSS gene probe, human HSS was amplified by RT-PCR. After DNA sequence analysis, a gene named hHSS containing an open reading frame (open reading frame, ORF) was obtained, which was inserted into the pcDNA3.1 plasmid (Invitrogen ) to construct pcDNA3.1-hHSS at the corresponding site.

Embodiment 2

[0029] Example 2: Cell culture and transfection

[0030] The BEL-7402 liver cancer cell line was cultured in DEME medium containing 15% fetal bovine serum at 37°C, 5% CO 2 cultivated under conditions.

[0031] Transfection: transfection and screening with hHSS gene by 1×10 4 Inoculate BEL-7402 liver cancer cells at a cell density of / ml in three 25cm 2 In culture flasks, cells were cultured in DMEM medium. When the cell density reaches 3×10 5 / ml (60-80% cell confluency), remove the culture medium, with no Ca 2+ , Mg 2+ Dulbecco's PBS (pH 7.4) washed 3 times to eliminate the influence of residual serum. Serum-free DMEM medium (containing insulin 10 μg / ml) was added. The hHSS gene transfection was carried out the next day. Prepare four 1.5ml sterile Eppendorf tubes, two corresponding to the marks. Take 5 μg each of pcDNA3.1-hHSS and pcDNA3.1, put them into two Eppendorf tubes, and make 0.1 μg / μl with HBS (HEPES-buffer saline). Another 60 μl of DOTAP (N-[2,3-Dioleoylox...

Embodiment 3

[0033] Example 3: Physicochemical properties and structural stability of recombinant hHSS

[0034] Under conditions suitable for recombinant protein expression, the monoclonal cell lines obtained in Example 2 expressing human recombinant liver stimulating factor and empty vector were expressed. The human recombinant liver-stimulating factor protein was recovered and purified, and its physical and chemical properties and structural stability were detected by synchronous fluorescence spectroscopy and circular dichroism spectroscopy.

[0035] see figure 1 , the test results showed that the purified recombinant hHSS was a homodimeric protein with a molecular weight of 31kD and an isoelectric point of 4.5.

[0036] see figure 2 3. Through the research of recombinant hHSS by simultaneous fluorescence spectroscopy and circular dichroism spectroscopy, it was found that the secondary structure of hHSS is very stable, and has strong acid and alkali resistance and high temperature res...

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Abstract

The invention relates to an application of a recombinant human hepatic stimulator factor in preparing medicines for restricting the apoptosis of tumor cells. After being purified, the recombinant human hepatic stimulator factor is a homologous dimmer protein with a molecular weight of 31kD, an isoelectric point of 4.5, the structure of which is stable with a pH value of 4-10 and a temperature of no more than 80 DEG C. The invention also relates an application of the recombinant human hepatic stimulator factor in restricting the apoptosis of tumor cells.

Description

technical field [0001] The invention relates to the effect of liver stimulating factor (HSS) on enhancing the anti-apoptosis effect of liver cancer cells and its application in the medical field. Background of the invention [0002] Hepatic stimulator substance (HSS) was first extracted by LaBrecque et al. in 1975 from the regenerated liver of partially resected rats, and found that it could specifically stimulate the proliferation of hepatocytes (1.Teir H, Ravanti K. Mitotic activity and growth factors in the liver of the whole rat. Exp Cell Res, 5:500-507, 1953). Later, the presence of HSS was found in livers of weaned rats and various animal livers including human livers. Studies have confirmed that HSS is a heat-stable, strongly negatively charged polypeptide substance with a molecular weight of about 15kD and has the characteristics of acid resistance and alkali resistance (Blomqvist K. Growth stimulation in the liver and tumor development following intraperitoneal inj...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/18A61P35/00
Inventor 安威吴媛陈莉阳志成杨琳杜海军
Owner CAPITAL UNIVERSITY OF MEDICAL SCIENCES