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Natural killer cell for transgenic expression cell chemotactic factors

A technology of natural killer cells and chemokines, which is applied in gene therapy, genetic engineering, plant gene improvement, etc., can solve the problem of not being able to reach the diseased part, and achieve the effect of increasing the killing probability, reducing loss, and improving the effect

Inactive Publication Date: 2005-06-22
龚小迪
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The main purpose of the present invention is to overcome the shortcomings of the currently developed cellular immunotherapy method that only a small amount of cell lines enter the blood circulation after injection, and most of the remaining cell lines cannot reach the site of the disease and disappear to the spleen and other organs. And provide a kind of natural killer cell that transgene expresses cell chemokine, it is that natural killer cell line transfers the specific chemokine, chemokine receptor conjugate or CCR10 of human tissue and organ, can reduce the loss of this cell line, Improve the effect of bioavailability of killing cells in vivo, so that more killing cell lines can reach the site of disease, that is, increase the number of effective parts, thereby effectively improving the therapeutic effect, reducing side effects caused by treatment, and fundamentally solving the key problems of clinical application

Method used

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  • Natural killer cell for transgenic expression cell chemotactic factors
  • Natural killer cell for transgenic expression cell chemotactic factors
  • Natural killer cell for transgenic expression cell chemotactic factors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1: Clone the human chemokine SDF-1 gene fragment and the SDF-1P2G gene fragment transformed from SDF-1 and construct the expression vector of the above fragments.

[0074] RNA was prepared from human vascular endothelial tissue according to the Trizol method. The mRNA was converted into a cDNA library by reverse transcription biochemical reaction. A set of primers was designed as follows: 5' primer, 5'-TTGCTAGCTCCACCATGAACGCCAAGGTC and 3' primer, 5'-TACAAGCTTGCCATCTTGAACCTCTTGTTTAA. This method was used to obtain the cDNA of the chemokine SDF-1 including the leader sequence. The cDNA of SDF-1 is cloned into a plasmid vector such as PCR2.1 vector (INVITROGEN). In order to obtain the nucleic acid sequence of SDF-1P2G, the gene of SDF-1 was transformed into the sequence ND by using the gene mutation method. 2, namely SDF-1P2G. Then, the above-mentioned SDF-1 and the transformed SDF-1P2G gene fragments are respectively connected to the expression vector through r...

Embodiment 2

[0077] Example 2: Transduction of natural killer cell lines with chemokine SDF-1 and ELC genes and their expression.

[0078] The specific method is as follows: linearize the DNA of the grafted chemokine gene vector, SDF-1-pcDNA3.1 and ELC-pcDNA3.1 plasmid vector with a restriction endonuclease, and transfer it into a natural killer cell line by electric shock In NK92 cells: prepare 1 x 10 7 NK-92 cells were resuspended in 10 μl of phosphate buffered saline and 90 μl of the solution provided by AMEXA. Add 5 µg of DNA and mix with cells. The carrier DNA was transferred into NK-92 cells by electroporation using AMEXA's electroporation instrument. Incubate at 37°C for 10 minutes and then switch to CO 2 Incubator for 4 hours. Since the vector has an anti-Gentamycin gene, Gentamycin was used to screen the expression of the vector. After 4 weeks of screening with Gentamycin (4 mg / ml), most of the cells died, and only a few cells survived. Thus, a stable expression cell line was...

Embodiment 3

[0080] Example 3: Comparison of the growth curves of the natural killer cell line transfected with the SDF-1 gene and the original natural killer cell line.

[0081] In order to determine whether the natural killer cell NK-92 with transchemokine gene is different from the original NK-92 cell line, the growth rate of the two cell lines of NK-92 transfected with SDF-1 gene and the original NK-92 was first compared Compare with the shape, such as figure 2 As shown, under the culture condition containing lymph interleukin IL-2, the growth rate of the two cell lines of NK-92 transfected with SDF-1 gene and the original NK-92 is basically the same, and divides about once every 24 hours. In the absence of Under the condition of lymph interleukin IL-2, neither the NK-92 of the SDF-1 gene nor the original NK-92 cell lines could grow and divide (using 3 H-TdR labeling test confirmed, the results are not shown here), this test shows that transfection of SDF-1 gene did not change the dep...

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Abstract

The invention provides a natural killer cell for transgenic expression cell chemotactic factors, which is the chemotactic factor expressing specific functions of human body tissue organs, chemotactic factor receptor bonder or CCR10, comprising the ligand chemotactic factor of the chemotactic factor receptor CXCR4, ligand chemotactic factor of CCR6, ligand chemotactic factor of CCR7, ligand chemotactic factor of CCR10, genes and protein sequence represented by sequence 1 and homologues and modification substance. The invention also provides the application of the cell strain in preparing medicament for treating tumor, virus or bacterial infection and autoimmune diseases, and its preparing process.

Description

1. Technical field [0001] The invention relates to a natural killer cell transgenicly expressing a cell chemokine, as well as its preparation method and application. 2. Background technology [0002] Malignant tumors are still the diseases with the highest mortality rate in my country. Once the tumor spreads and metastasizes, no surgery, chemotherapy or radiotherapy can completely eradicate the tumor in the body. Tumors that have spread metastatically eventually grow malignantly, leading to the death of the patient. So far, there is no real way to prevent and treat tumor metastasis. On the one hand, these treatments are limited by conditions and doses. The side effects caused by excessive radiation therapy or chemotherapy make the patient unbearable and even die. On the other hand, many tumors mutate continuously during treatment, making them less sensitive to drugs and radiation. Studies have found that most of these drug-resistant and radiation-resistant tumor cells a...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61P31/00A61P35/00A61P37/00C12N5/08C12N5/10C12N15/12
Inventor 龚疆红
Owner 龚小迪
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