| IntroductionLow back pain and leg pain is a more common disease in orthopaedics. Many mechanical factors and inflammatory factors affect the process of lumbar disk herniation. Recently, researchers did many studies about the effect of interleukin to the metabolism of proteoglycan in the cartilage matrix of lumbar intervertebral disc. The studies show that small dose interleukin - 1 can restrain the synthesis of proteoglycan in the cartilage matrix, however, large dose interleukin - 1 stimulate the degradation of proteoglycan. The results indicate interleukin -1 is important to the metabolism of cartilage matrix. Interleukin - 1 has two kinds; IL - α and IL - 1β. Studies show IL - a to the degradation of proteoglycan in the matrix have time - and dose - dependency , but about IL - β hasn' t similar reports. This investigation is to disclose the relation between IL - β and proteoglycan metabolism through the culture of fetal intervertebral disc.Materials一, Object and groupThe fetus of 34 weeks obtained from spontaneous abortion was used in this study. The intervertebral disc was taken and cultured invitro, within 4 hours after abortion. 二, Principal reagent1. IL - 1a; IL -1 b(Pepro tech EC Ltd company)2. D - Hanks solution3. 8mol/L guanidine hydrochloride4. DMSO eluent5. Triton -1006. Alcian solution ( Alcian 8GS chroma - gesellschaft, Kongen, Germany company)7. DMEM -5% FBS culture medium 三, Principal instrument1. High speed centrifugation instrument2. 96 wells color matching broad3. 37 Ti CO2 incubator4. EL 800 enzyme - labelled instrument ( Bio - TEK UBSTRYNEBTS. INC)Methods1. The fetus of 34 weeks obtained from spontaneous abortion was used in this study. The intervertebral disc was taken within 4 hours after abortion and washed by buffer solution, then took out nucleus pul-pous and dip into D - Hanks solution. Separate the nucleus pulpous into pieces of 2mm diameter and 1mm high. Cultured the pieces in 25ml DMEM -5% FBS culture medium. In 37C CO2 incubator cultured 2 days. Transported the specimens to 96 wells culture broad filled 150ul DMEM culture medium.2. Different effect of different concentration IL - 1aand IL - lbtoPG in the nucleus pulpous; Marked 42 wells from 96 wells culture broad and divided into 6 groups. Respectively add in IL - la and IL - lp 0.1 ,0. 2,0. 4nmol/L. Cultured 30 hours in 37C 5% CO2 incubator, determinate the concentration of proteoglycan in the culture medium.3. The effect of IL - 1b to metabolism of PG though different culture time: Marked 36 wells from 96 wells culture broad and divided into 3 groups, then group 6 matches each group. Distributed randomly control group and experiment group. Respectively added in IL - 1b 0. 3nmol/L. In 31C 5% CO2 incubator cultured 12,24,72 hours and respectively determined concentration of chondroitin sulfate at different time.4. Detection methods for chondroitin sulfate: Took 50u,l sample and added in 8mol/L 50ul guanidine hydrochloride, after 15 minutes added 50ul reagent 1 (high concentration Alcian solution) , agitating until completely dissolve. Then added 750ul reagent 2B (low concentration Alcian solution) , cultured 1 hour in 4C condition, discarded supernatant solution after centrifugated 15 minutes with 12000r/min; added 750ul DMSO eluent, stirred 30 minutes in room temperature, discarded supernatant solution after centrifugated 15 minutes with 12000r/min again. Added 500 ul lysis liquor, stirred 15 minutes in room temperature. Took 240 ul two samples liquor from the same sample liquor and put the wells of the 96 wells color matching broad, determined concentration of chondroitin sulfate with enzyme - labelled instrument under 630nm wave - length.Results一,The effect of IL - la and IL - 1b to degeneration of proteo-glycan in matrix of nucleus pulposus. It was found that IL - la and IL - 1 (3 can stimulate degeneration of proteoglycan after 30 hours culture. The difference of concentration of chondro...
|
PDF Full Text Request