Effect Of The Derivitives From Trypsin Digested ConA On The Proliferation And Differetiation Of Cultured Rabbit Resting Condrocyte

Deformation arthritis, degenerative joint disease and injury of themechanical joint gradually increase in the incidence of disease.Because of the special nature of the cartilage nature, which is lackingin nutrition supplication by blood vessels, lymph, and nerve system,great difficulties are caused in the treatment of the cartilage disease.Using vitro cultured condrocyte in the cartilage organizationengineering , the key to rehabilitation of cartilage damage has becomea hot study in this field all over the world. Cartilage cell is one of theearliest seed cells applied in the organization engineering, but becauseof its source, its multiplication capacity is limited and the long-termvitro cultivated cartilage cell is prone to dedifferentiation and loss ofbiological activity of cartilage cell, its application is gradually limited.Therefore the research on the proliferation and differentiationmechanisms of cartilage, and the search of powerful stimulus bringgreat help to the development of cartilage organization engineeringand the prevention and cure of cartilage disease.Concanavalin A (ConA) is plant agglutinant separated andpurified from the great sword bean for the first time in 1919 bySumner. Later, Sumner and Howell found that the purified ConAcould not only agglutinate red blood cell, but also sedimentate thesugar and starch in the liquid. Along with the development ofresearch on ConA, its biological activity is gradually recognized.ConA has a strong effect on promoting lymphocyte mitosis andstimulating lymphocyte multiplication. It can activate humanlymphocyte cells into lymphatic mother cells, thereby strengtheningimmunity. It is capable of agglomerating cancer cells anddeformation cells resulted from various carcinogenic substances, but ithas no side effect on normal cells, therefore it is able to be used toanti-tumor. It is also able to activate inhibited T cells selectively, andplays an important role in regulating the organisms immune response,so the way through the use of ConA to activate T cells is expected tomeliorate some of their own immune inhibited diseases or even organtransplant rejection response, and have good prospects in theimmunization and treatment of malignant neoplasm.ConA exerts great impact on promoting differentiation ofcartilage cells. It can stimulate and promote the cell synthesisALPase , X collagen, compounding of VD3R and calcification ofmatrix of cultivated resting cartilage cells. Although divalentSuccinyl-ConA can not bring out any change of cartilage cell skeletonor restrain the cell expansion, it has similar effect with the tetravalentConA to stimulate ALPase activity of cartilage cell, Agreaconsynthesis, the total protein synthesis and inhibite DNA synthesis.This is different from the observation of vitro cultivated lymphocyte.ConA can change the lymphocyte shape and stimulate themultiplication of lymphocyte at the same time, whereasSuccinyl-ConA can not change the lymphocyte shape and no effect isobserved in lymphocyte proliferation. Therefore we can reach aconclusion that the regulatory role of ConA is achieved at leastthrough two regulatory systems in congdrocyte and lymphocyte.Currently, the find of two ConA cell receptors also confirmed theregulatory role of ConA is achieved at least through two regulatorysystems in congdrocyte and lymphocyte in another aspect.MTf (membrane-bound transferrin like protein) is initially a kindof 97Kda antigen related to the tumor which is firmed through atellin.It has same aminophenol sequence (~40﹪) with transferrin andlactoferrin. MTf has potential to N-link sugar basis and can beadsorbed on ConA-Sepharose column.Both ConA and Succinyl-ConA can be mixed with MTf toregulate cartilage cell and ATDC5 cartilage polysaccharide synthesis,DNA synthesis, and the expression of collagen II . Anti-MTf atellincan eliminate the effect of ConA on the cartilage cell. FurthermoreMTf is not expressed in all cells but only in a few cells, including thecartilage cell.Another surface receptor combined with ConA, PZR is awidely-expressed member in the super immune globulin family. It isexpressed in a variety of blood cells. Its intracellular segmentcontains two immunoreceptor tyrosine-based inhibition motifs (ITIM),which can be compounded with the specificity of SHP-2【43】 afterphosphorylation. SHP-2 is phosphatase containing SH2 structure andit plays an important role in signal path in the cell. When ConAfunctions, it causes the gathering of PZR, mutual phosphorylation oftyrosine spaces point among molecules, and then a combination withSHP-2, which takes the role of tyrosine enzyme. Then PZR isphosphorylated and completes the initial phosphorylation and signaltransmission. Succinyl-ConA does not change the lymphocytepattern or increase the lymphocyte multiplication probably because itcannot be conglomerated with PZR.ConA as the plant-originated stimulus carries on great effect inpromoting defferentiation of cartilage cell. Although it does not havethe nature of human immune, it has a variety of different biologicalactivity to varied cells, its function mechanism and target are not yetclear. This point seriously affects the application of ConA in thecartilage organization project and clinical treatment.Therefore, in our experiment, we control digestive conditions bybreaking ConA into small fragments and then use Mono S ionexchange Chromatography to separate and purify them. Finally weget a small segment such as 18Kda is called C18K.Then we put theminto the vitro cultivated cartilage cell and check the effect of digestedproduct C18K. Then we expect to make clear the active domain ofConA and make its effect exclusive, which lays foundation for theclinical and cartilage research to.Ⅰthe digestion of ConA and the separation and purification ofdigestive products.We control the digestive conditions of trypsin and digest ConAfor three hours. ConA is fully digested. By using Mono S ionexchange Chromatography a group marked with C18K is separated.SDS-PAGE is identified to get a 18kDa electrophoresis band esxceptthe band of trypsin.II The cultivation of cartilage cell of original generationChondrocytes were isolated from the growth plate and restingcartilage of ribs of 4-week-old male Japanese White rabbits, andwere seeded at 104 cells/6-mm plastic microwell, 105 cells/cell of 24cells plank and grown in 0.1 ml or 1ml of DMEM mediumsupplemented with 10% fetal bovine serum.Change the medium every 4 days.III testing of cartilage cell multiplication1 MTT measuring cell multiplicationWhen 80% of cells are confluence, change testing cultivatemedium, the contral group being 2%NBCS DMEM cultivate fluid, andadd DMEM containing 10μg/mlConA and 20μg/mlC18K to thepositive comparison and testing groups respectively.Measure the MTT indicator of cell and map the growth curve onthe third, fifth, seventh and tenth day after adding testing cultivativeliquid. It is obtained that the cells in the contal group have normalvitro growth, consistent with cartilage cell multiplication circle. Thecells in the testing group grow slowly and stop growing after addedConA or C18K.2 ALPase activity testingUse ALPase testing kit to measure the enzyme activity on thethird, seventh, tenth, fifth and twentith day after adding testingcultivative liquid. As it can be seen, in the testing group and positivecomparison group the enzyme activity is remarkably improved, muchhigher than the contral group. It is statistically significant.3Alizarin red stainingFix the cells and stain them with Alizarin red dyeing on theseventh, fiftith, twenty-first and thirtith day after adding testingcultivative liquid. The result is that positive reaction appears in thetesting group and positive comparison group from the twenty-first day,while negative reaction appears in the contral group.To be concluded, the active segment C18K derived from ConA inthe experiment can forcefully inhibite the multiplication of thecartilage cell, and promote the cell defferentiation. To explore thebiological function of ConA protein exactly, the obtainment ofexclusive active segment provides the preconditions for the cartilageorganization engineering studies and clinical applications.