| Objective: To provide experimental foundation for clinicalpractice of gene therapy of disc degeneration disease byobserving the expression of hTGF-β1 gene whichtransferred the degenerated intervertebral disc of mature NewZealand rabbits.Methods1.To build the animal model: 24 mature New Zealandrabbits of 6-month age were chosen, regardless of sex, whichwere average for 2.5 kilograms. To enter the right-side oflumber vertebrase and expose intervertebral disc from theexterior of peritoneum. Then injected 0.02ml N.S. intodifferent intervertebral discs (L3-4, L4-5, L5-6, L6-7) . Afteroperation, all rabbits were being fed in different cages fortwelve weeks. It all can be used to observe the modelbuilding state through X-ray and pathological diagnosis.2.To proliferate the recombinat plasmid vector: ①Toproliferate the E.coli JM109 germ in the flat panel in ahothouse of 37℃with the method of painting for a night,then choice a single germ and put it into the liquid of agar in37℃, shaking them to OD260=0.3～0.4. ②To make the germinto the sensitive state with the method of Cacl2, then addedinto it with the plasmid which contained the purpose gene. Toconvert the plasmid into the body of the germ by the methodof 42℃hotshock and continue to proliferate them overnight.③To spread the germ liquid to the flat panel which containedthe antibiotics and the X-gal, IPTG, then picked the neededgerm by the method of antibiotics and the experiment of theblue-white plot. After it , to proliferate the germ liquidovernight. ④To screen, purify the plasmid by the method ofcentrifuge, and to measure the density and the purity by theluminosity photometer, then to determine it with theinscribed ferment and the agar gel electrophoresis.3.To transfer invivo: to operate on 20 rabbits withsuccessful product model again, then enter the left–side oflumber vertebrase and expose the intervertebral disc lied inL5-6, L6-7, where it was injected 0.02ml the recombinateplasmid DNA carrying hTGF-β1 gene, as experimentalgroup. To inject the 0.02ml N.S. into L3-4, L4-5 , as controlgroup and L1-2, L2-3was as self-control group. After thesecond operation, all rabbits were fed in different cages. Eachtime 4 rabbits were killed until 4 days, 1 week, 2 week to 3week. The material would be attained from rabbits after thesecond operation. To make analysis the expression ofpurpose gene at mRNA and protein level by RT-PCR andwestern-blotting.4.Remedy results of hTGF-β1: to kill the left 4 rabbitsafter 4 weeks of the second operation, attaining material tomake up pathology slice in order to observe the situationwhich hTGF-β1 makes effect on reserving the degenerationof the intervertebral disc.Results1. To identify the animal model: ①Observing by eyes:after 12weeks of making model operations, to kill 4 modelanimals at random, it can be found that the edge of theintervertebral disc(L3-4, L 4-5 , L5-6, L6-7) and the contiguoustissue were hardly separable. The intervertebral disc were incalcification(the 7 code needle can't pierce to theintervertebral disc). The origins of the nerves wereinseparable.②To observe by X-ray: after 12 weeks, theintervertebral space become narrow obviously, thecalcification of intervertebral space can be foundoccasionally. ③To observe in the microscope: fibers lineddisorderly, rupture, small gap can be found occasionally. Thenucleus pulposus cells diminished and occurredprogrammed-death and necrosis.2. To determine the proliferation of the recombinatplasmid: ①To measure the density and the purify by theluminosity photometer: (the density of the nucleicacid=OD260×the times of the dilution×50/1000, the purifyof the nucleic acid=OD260/OD280=1.6～1.8). According to theformula, to observe that the density of the nucleic acid is0.067 ug/ul and the purify of the nucleic acid is 1.782. ②Todetermine the recombinat plasmid: to make use of theinscribed ferment and the agar gel electrophonesis and at lastmake a conclusion that the recombinant plasmid contain thegene of hTGF-β13. The analysis of the expression of purpose gene : Toanalyze the expression of purpose gene in various periods ofthree groups by methods of RT-PCR and Western-blotting atthe level of mRNA and protein, through software of makingpicture Bio ID technology in Vilber Lourmat CompanyFrance. The results showed: ①experimental group: theresults of RT-PCR indicated that hTGF-β1 mRNA can bedetected , but less than self-control group 4 days aftersecond-operation. It sustained clamix until 1 week aftersecond-operation, the expression of hTGF-β1 mRNA issignificantly higher than control group and self control group.The expression of hTGF-β1 mRNA began to decline, but ishigher than control group and self control group 2 weeksafter second-operation, which remained declining, butmaintain higher level after 3 weeks of second-operation .Western-blotting showed that hTGF-β1 can be found whileexpression level is very low after 4 days of second-operation.The expression of protein level has begun to raise 1 weekafter second-operation, then come to clamix till 2 weeks aftersecond-operation, obviously higher than control group andself control group(This is significant difference). It began todecline and it is higher than control group and self controlgroup 3 weeks after second-operation(This is significantdifference). It showed that hTGF-β1 gene has begun toexpress through injecting recombinat plasmid vector DNAcarrying purpose gene in experimental group. ②Thecontrol group: The results of RT-PCR showed: the expressionof hTGF-β1 mRNA was at lower level in various periods,change maintained relative steadiness which was much lowerthan experimental group and self control group(This issignificant difference). The results of Western-blottingindicated that the expression of hTGF-β1 proteinmaintained relative steadiness which is lower thanexperimental group and is higher than self control group(Thisis significant difference). ③The self control group: Theresults of RT-PCR showed that the expression of hTGF-β1mRNA was relatively steady in vavious periods, which washigher than control group. The results of Western-blottingindicated that the expressin of hTGF-β1 protein maintainedrelative steadiness which is lower than experimental groupand control group.4. The results of hTGF-β1 gene remedy: to kill the left 4... |
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