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Treatment on human malignant solid tumor by HNF4 alpha inducement and differentiation

A solid tumor, alpha protein technology, applied in the fields of cell biology, medicine, and molecular biology, can solve the problems of unscreened drugs or proteins for differentiation therapy, inability to differentiate tumors, and reducing the tumorigenic effect of cancer cells.

Active Publication Date: 2009-09-09
上海赛迪夫医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, although it has been confirmed that some substances or genes can improve certain biological characteristics of tumor cells in vitro experiments (such as reduced proliferation and clone formation ability, up-regulated expression of some normal cell function genes), some substances have even been proved to reduce cancer cells. Tumor formation of cells in animals, but it is often found that these substances also affect normal cells (side effects), and cannot specifically induce differentiation of solid tumors in vivo
The present inventors have studied the regulatory effects of all-trans retinoic acid, somatostatin, tumor necrosis factor, and arsenic trioxide on the differentiation of liver cancer cell lines in vitro, but no drugs or proteins with definite therapeutic effects on differentiation have been screened out. These substances were also found to be ineffective in inducing differentiation of solid tumors

Method used

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  • Treatment on human malignant solid tumor by HNF4 alpha inducement and differentiation
  • Treatment on human malignant solid tumor by HNF4 alpha inducement and differentiation
  • Treatment on human malignant solid tumor by HNF4 alpha inducement and differentiation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] RT-PCR detection of expression of HNF4α gene and hepatocyte-related functional genes in human liver tumor cell line

[0082] 1. The commercially available conventional liver tumor cell lines Huh-7, Hep3B, and HepG2 were all mixed with 8×10 5 Inoculated in a six-well plate per plate, cultivated with fresh culture medium containing 10% fetal bovine serum, extracted cellular RNA the next day, and measured OD with a spectrophotometer 260 Values ​​were formulated into working concentrations (1 μg / μl and 0.1 μg / μl), and RNA integrity was detected by 1% agarose gel electrophoresis.

[0083] 2. RT-PCR: Take 4 μg RNA, 2 μl random primers, add DEPC water to 33 μl, set at 70°C for 5 minutes, and set at 0°C for 5 minutes, then add 10 μl 5×Buffer, 3 μl dNTP, 2 μl RNA reverse transcriptase and 2 μl RNase inhibitor After mixing, place at 37°C for 1.5h to obtain the reverse transcription product. After diluting the reverse transcription product, 1 μl was taken as a template for PCR a...

Embodiment 2

[0092] Construction of recombinant replication-defective adenovirus AdHNF4α expressing HNF4α

[0093] 1. Obtaining the HNF4α 1425bp cDNA fragment: Design and synthesize primers according to the human HNF4α cDNA sequence. Sense primer (Bgl II restriction site added at the 5' end): 5'-CCG AGA TCT AGA ATGCGA CTC TCC AAA ACC-3' (SEQ ID NO: 21). 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: 22).

[0094] 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 harvested and placed in an Eppendorf tube, and the weight of the gel was weighed. Add 200ml of NT solution / 100mg gel into the Eppendorf tube, incubate at 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...

Embodiment 3

[0100] RT-PCR and Western blot detection of HNF4α expression after AdHNF4α infection of human liver tumor cell lines

[0101] 1. Both human liver tumor cell lines HepG2 and Hep3B were treated with 5×10 5 / dish was inoculated in a six-well plate, and the virus AdHNF4α was infected with MOI 40 and 100 respectively. After 24 hours, the fresh DMEM medium containing 10% fetal bovine serum was replaced, and GFP expression was observed after 3 days ( Figure 6 ). Total RNA was extracted with the Trizol kit, reverse transcription was performed for 2 hours, and 1 μl of the diluted reverse transcription product was used as a template for HNF4α PCR amplification. The reaction system is as follows.

[0102]

[0103]The reaction conditions were 95°C for 30s, 55°C for 30s, 72°C for 90s, 27 cycles.

[0104] The RT-PCR products were identified by 1.5% agarose gel electrophoresis, and after the image was scanned, densitometric scanning and sequencing analysis were performed with Multy-An...

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Abstract

The invention relates to treatment on human malignant solid tumor by HNF4 alpha inducement and differentiation, in particularly to a method for utilizing Hepatocyte Neclear Factor-4 alpha (HNF4 alpha) to induce the differentiation of the human malignant solid tumor so as to be applied to the treatment on the malignant solid tumor and application thereof. The research shows that the modulation of gene expression of the HNF4 alpha of malignant solid tumor cells can effectively produce functions of the inducement and the differentiation on the tumor cells so as to provide a new treatment means for inducing and differentiating the tumor.

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 use of using Hepatocyte Nuclear Factor-4α (Hepatocyte Nuclear Factor-4α, HNF4α) to induce differentiation of human malignant solid tumor cells, thereby applying it to the treatment of solid tumors. Background technique [0002] Tumor differentiation therapy (differentiation therapy) is an important breakthrough in the clinical treatment of tumors in the past 20 years. Induced differentiation therapy is to promote the differentiation of tumor cells to mature stage by inducing differentiation method, restore normal cell phenotype and function and inhibit the proliferation of malignant tumor cells. Differentiation-inducing therapy breaks the traditional understanding that tumor development is irreversible, and strongly promotes the development of the entire cancer research field. [0003] Chinese scholars hav...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/19A61K48/00A61K9/08A61P35/00
CPCA61K38/1709A61P35/00
Inventor 谢渭芬尹川林勇陈岳祥曾欣
Owner 上海赛迪夫医药科技有限公司
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