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Gene engineering medicine for curing diseases relative to vascularization

A new blood vessel and disease technology, applied in the field of disease medicine, to achieve high yield, low pollution, and the effect of inhibiting the growth of blood vessel cells

Inactive Publication Date: 2003-04-09
HUAXINGSHENG BIOLOGICAL TECH BEJING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the use of Plasminogen K5 as a drug for the treatment of neovascularization-related tumors and retinal diseases has not been reported yet.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The preparation of embodiment 1 K5

[0033] 1. Construction of human recombinant K5 expression system

[0034] K5 cDNA was amplified from human liver RNA by RT-PCR (reverse transcription polymerase chain reaction), and the PCR product was cloned into the Eco RI and Hind III positions of the pET22 vector (product of Novagen, USA). At this point, a fusion gene sequence of 6X histidine and K5 was formed. The correctness of the expression vector construction was confirmed by sequencing.

[0035] 2. Expression and purification of human recombinant K5

[0036] The above vector was transformed into E. coli strain BL-21 / DE3 (product of Novagen, USA). The human recombinant K5 expressed by this strain is mainly in the space of the plasma membrane (preplasmic). The expression of the recombinant protein was induced by IPTG. The protein in the plasma membrane space was recovered by lysozyme and centrifugation. The protein thus obtained was separated and purified through a His-...

Embodiment 2

[0037] Example 2 K5 inhibits growth of human retinal cells

[0038] 1. Experimental materials

[0039] Source of retinal cells: volunteer human eyes were obtained from the South Carolina Lion's EyeBank Association.

[0040] 2. Experimental method

[0041] A. Isolation and culture of human retinal capillary endothelial cells (HRCEC) and pericyte:

[0042] Retinal capillary endothelial cells (HRCEC) isolated from human eyes incorporated acetylated LDL (DiI 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanineperchlorate, Biomedical Technologies Inc. , Stoughton, MA), to determine the purity of the second passage endothelial cells in culture. Pericyte was isolated according to the literature (Grant MB, Guay, C: Plasminogen activator production by human retina endothelial cells of non-diabetic and diabetic origin. Invest Ophthalmol Vis Sci 32:53-64, 1991) method.

[0043] B. Viable cell count:

[0044] The above-mentioned cells in the second and sixth passages were seeded in a...

Embodiment 3

[0049] Example 3 Cell type-specific inhibition of K5

[0050] The experimental conditions were the same as in Example 2, and the results showed that K5 did not show any significant inhibitory effect on primary pericytes or E1A-NR3 cells (an immortalized cell line derived from rat retinal neurons) (p>0.05, n=4). This was observed for concentrations of K5 ranging from 20-320 nM. Tip: The inhibitory effect of K5 is only for neuroendothelial cells.

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Abstract

The present invention relates to a kind of medicine containing human recombinant plasminogen K5 segment and for treating diseases relative to vascularization. Recombinant K5 is used in the experiment of human eye retina capillary endothelial cell growth inhibition, the in vivo experiment of preventing and inhibiting rat retina nerve vascularization and the experiment of K5 antitumor. The results show that recombinant K5 has obvious in vivo and in vitro inhibition on retina vascularization and can inhibit tumor growth obviously while has no poison and damage to non-vascular cell.

Description

Technical field: [0001] The invention relates to a genetic engineering drug, in particular to a drug containing human recombinant plasminogen K5 fragment for treating diseases related to neovascularization. Background technique: [0002] Angiogenesis is the result or factor of a complex physiological and pathological process, which can occur under normal physiological conditions (such as wound healing, pregnancy and development) and pathological conditions such as retinal hyperemia and tumorigenesis caused by diabetes. related to the formation of new blood vessels. In particular retinal neurovascularization, the abnormal formation of new blood vessels from preexisting capillaries, is a characteristic pathological phenomenon of many eye diseases. Such as diabetic retinopathy, sickle cell retinopathy, retinal vessel occlusion and retinal prematurity (ROP). Tumors, cerebral ischemia, and chronic inflammation can also lead to neovascularization, thereby exacerbating these cond...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/43A61P27/02A61P35/00
Inventor 顾军马建新
Owner HUAXINGSHENG BIOLOGICAL TECH BEJING
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