Method for detecting bioactivity factor reserved in acellular matrix

A technology of biologically active factors and cell-free matrix, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of unclear adhesion factors and chemokines, single detection of growth factors, etc. The effect of prolonging the inactivation time

Inactive Publication Date: 2008-12-24
THE AFFILIATED DRUM TOWER HOSPITAL MEDICAL SCHOOL OF NANJING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the cell proliferation assay can only detect the growth factors retained in the cell-free matrix, and the adhesion factors and chemokines that play an important role in the repair and regeneration of tissues and organs are still unclear.

Method used

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  • Method for detecting bioactivity factor reserved in acellular matrix
  • Method for detecting bioactivity factor reserved in acellular matrix
  • Method for detecting bioactivity factor reserved in acellular matrix

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1 The preparation of porcine bladder cell-free matrix, the preparation steps are as follows:

[0075] a) Acquisition of pig bladder: Take out all the bladder and part of the urethra of a pig weighing 20 kg, put them in 4°C pre-cooled 10mM phosphate buffer solution, pH 7.2-7.4, quickly bring them to the laboratory and carefully remove them Except for fat and serosal tissue on the surface, insert an infusion thong catheter through the urethra and fix it on the urethra with silk thread, and then flush the bladder with 4°C pre-cooled 10mM phosphate buffer, pH 7.2-7.4, through the infusion thong catheter 3 times to wash out the urine in the bladder cavity;

[0076] b) Pour 100ml of digestive juice containing 0.25% / 0.038% trypsin / tetrasodium edetate into the bladder cavity through the infusion thong catheter, the pH is 7.2-7.4, clamp the infusion thong catheter, and then place the entire bladder Completely immerse in the digestion solution, shake and digest at room t...

Embodiment 2

[0089] The preparation of embodiment 2 porcine small intestine submucosa, its preparation steps are as follows:

[0090] a) Acquisition of pig small intestine: Take a section of pig small intestine with a length of about 20 cm, wash it with 10 mM phosphate buffer (PH7.2-7.4) pre-cooled at 4°C, and take it to the laboratory;

[0091] b) Carefully cut off the mesentery with ophthalmic scissors, carefully scrape off the adventitia and muscular layer of the small intestine with a scalpel, then turn the small intestine so that the mucosal layer of the small intestine faces outward, and scrape off the epithelial layer and lamina propria of the mucosa in the same way to obtain a thick layer. Translucent film of about 0.1mm;

[0092] c) Place the above-mentioned translucent film in 40ml of 50mM tris-hydrochloric acid buffer solution containing 0.2% Triton X-100, pH 8.0, containing 0.5M sodium chloride, 10mM ethylene dichloride Tetrasodium amine tetraacetate and 10KIU / ml aprotinin, sh...

Embodiment 3

[0102] Example 3. Isolation, cultivation and identification of seed cells

[0103] 3.1 Isolation, culture and identification of HBSMC

[0104] Patients with bladder cancer who underwent radical cystectomy agreed to take a small piece of bladder tissue without obvious tumor growth, and remove the urothelial layer completely by mechanical separation, using trypsin / ethylenediamine at a concentration of 0.25% / 0.038% Tetrasodium tetraacetate and 0.1% type I collagenase were sequentially digested, and the resulting cell suspension was inoculated in DMEM / F-12 full medium containing 10% FBS for primary culture and subculture. Inverted microscope was used for morphological observation, and the expression of smooth muscle actin and desmin was observed by immunostaining of cell slides.

[0105] Observed under an inverted microscope, the human bladder smooth muscle cells cultured in vitro were long-spindle-shaped, and showed a typical "peak-valley" shape after fusion; both smooth muscle ...

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Abstract

The invention discloses a method for detecting bioactive factors reserved in acellular matrix. The method of the cell biology is adopted to detect adhesive factors, growth factors and chemotactic factors reserved in the acellular matrix. The acellular matrix is subject to homogenate processing, the clear solution is absorbed to wrap a culture plate to do the adhesive experiment of seed cells in order to detect the adhesive factors reserved in the acellular matrix; the condition culture medium without the acellular matrix is prepared, the growth rate of the seed cells, the proliferation and the transportation activity, and the potential for healing the scratch are observed in the culture medium in the condition is observed in order to detect the growth factors and the chemotactic factors reserved in the acellular matrix. The method has an eye to the cytobiology effect of the bioactivity factors reserved in the acellular matrix, has simple and convenient detection technology and low in cost, and can be popularized and applied further.

Description

technical field [0001] The invention relates to a method for detecting biological materials, in particular to a method for detecting biologically active factors retained in acellular matrix. Background technique [0002] At present, acellular matrix is ​​a biomaterial that has been studied and applied more, and has shown great prospects for clinical application, such as small intestinal submucosa (SIS) and bladder acellular matrix (BAM). The researchers used urea, guanidine hydrochloride, etc. to extract the total protein in SIS and BAM, and used molecular biology techniques, such as Western blot (WB), immunohistochemistry (IHC), to qualitatively detect growth factors, and enzyme-linked immunosorbent assay. Assay (ELISA) was used to quantitatively detect growth factors, and the extracts were also used in cell proliferation experiments to observe their biological activities. The results showed that there were growth factors such as VEGF, bFGF and TGF-β in SIS; porcine bladde...

Claims

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Application Information

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IPC IPC(8): C12Q1/02
Inventor 杨斌孙则禹戴玉田
Owner THE AFFILIATED DRUM TOWER HOSPITAL MEDICAL SCHOOL OF NANJING UNIV
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