The Experimental Study On Prevention Of Deep Vein Thrombosis By LRRFIP1Gene Interference In Orthopedic Surgery

BackgroundDeep venous thrombosis （DVT） is one of the common complications in the departmentof orthopedics, occuring in the patients with fracture of lower limb. If the thrombi fall off, itcan cause fatal pulmonary embolism, and often left phlebitis syndrome, which seriouslyaffect the rehabilitation and life safety of patients. Recent studies have shown that leucinerich repeat interacting protein1（LRRFIP1） may have important links with thrombosis.LRRFIP1gene and its encoded protein, may interact with the protein on platelet membrane,then change the platelet cytoskeleton structure, and affect the coagulation function inplatelet and thrombus formation. So we proposed to observe the effect of LRRFIP1gene onthe formation of DVT through silencing LRRFIP1by RNA interference, investigate theeffect of LRRFIP1gene silencing on the prevention of DVT and provide the experimentalbasis for the final application of gene therapy in the treatment of DVT in vivo.Methods1. Construction of lentiviral vector carrying LRRFIP1shRNADesign and synthesize LRRFIP1gene targeted double-strand DNA: according to therat LRRFIP1gene sequence reported by GenBank, using the design software of Ambioncompany, according to RNA interference sequence design principles, design3againstLRRFIP1gene shRNA oligonucleotide sequence. Construct and transfect the plasmids into293T cells, RT-PCR and Western blot methods determine the silencing efficiency. Themost efficient shRNA plasmid was used to package the lentiviral vectors.2. Isolation and culture of the primary mouse bone marrow cells （BMCs）One-week old rats were sacrificed after disinfection. BMCs were isolated andincubated in37C,5%CO2incubator. P3cells were selected for the followingexperiments. 3. Infection of BMCs with lentivirusBMCs were seeded in24-well plates at a density of3⁵X104/well. When theconfluence reached70%, the BMCs were infected with the lentivirus. After96h, thefluorescence was observed under the fluorescent microscope, and the infection efficiency ofthe lentivirus was estimated.4. Animal grouping and treatmentHealthy adult male ICR mice （20±2g） were randomly divided into the followinggroups:1) normal control group （control）;2) the sham operation group （sham）;3) lowmolecular weight heparin （LMWH） treatment group （LMWH）: mice were injected withLMWH (2000U kg^-1) once by tail vein preoperatively, and postoperatively injected withLMWH (2000U kg^-1 d^-1) daily;4) model group （model）: mice were injected with normalsaline daily;5) LRRFIP1shRNA treatment group （shRNA）: mice were injected with BMCsinfected with LRRFIP1shRNA lentivirus before operation （1×10⁷cells/mouse）;6)negative shRNA treatment group （Negative shRNA）: mice were injected with BMCsinfected with negative LRRFIP1shRNA lentivirus before operation （1×10⁷cells/mouse）.DVT mouse model was produced by ligation of inferior vena cava. Six mice in eachgroup were sacrificed on the1st, the3rdand the7thday post operation. The weight ofthrombus was determined; enzyme linked immunosorbent assay （ELISA） detection wasused to examine serum P-selectin and d-dimer levels; expression of LRRFIP1protein inthrombosis was determined by Western blot.5. Statistical analysisMeasurement data were expressed as means±S.D. SPSS17for Windows was usedfor statistical analysis. Statistical analysis was programmed by one-way analysis ofvariance （ANOVA）. Difference was regarded as significant when P <0.05.Results1. The lentiviral vector carrying LRRFIP1shRNA was successfully constructed.transfection of LRRFIP1shRNA significantly decreased LRRFIP1mRNA and proteinexpression;2. LRRFIP1shRNA significantly inhibited the expression of LRRFIP1protein inthrombus. In the model group, the expression of LRRFIP1in thrombus decreasedsignificantly （P<0.01） on the1st,3rdand7th days after operation. At the3time points observed, LRRFIP1shRNA treatment further suppressed LRRFIP1expression in thrombus,indicating LRRFIP1shRNA lentivirus can effectively inhibit the LRRFIP1expression. Butthe LMWH and negative shRNA had no effect on the expression of LRRFIP1.3. LRRFIP1shRNA significantly inhibited mice DVT formations. LRRFIP1shRNAinhibited thrombus formation in vivo apparently. On the7th day post operation, the weightof thrombus in model group was8.63±0.40mg, whereas the thrombus weight reduced to2.15±0.57mg （P<0.01） in the LRRFIP1shRNA treatment group, with a thrombosisinhibition rate of75.10%. In the LMWH treatment group and shRNA negative group, thethrombus weight were2.20±0.33mg and6.47±0.66mg, with thrombosis inhibition ratesof74.50%and25.02%respectively.4. LRRFIP1shRNA can reduce the serum levels of p-selectin and d-dimer. Comparedwith the normal control group and sham operation group, the plasma level of P-selectinsignificantly increased （P<0.01） in model group,. LRRFIP1shRNA treatment significantlydecreased the plasma level of P-selectin （P<0.01）. The plasma level of d-dimer in modelgroup was also significantly increased （P<0.01）, and LRRFIP1shRNA treatmentsignificantly inhibited the up-regulation of d-dimer levels （P<0.01）. However, LMWH andnegative shRNA showed little effect on plasma P-selectin and d-dimer levels.Conclusion1. The LRRFIP1shRNA expression plasmid and lentiviral vector were successfullyconstructed;2. LRRFIP1shRNA is able to inhibit LRRFIP1mRNA and protein expression in vitroand in vivo;3. LRRFIP1gene knockout inhibits deep venous thrombosis formation in mice;4. Inhibition of deep vein thrombosis with LRRFIP1shRNA is associated with thedecrease of plasma p-selectin and d-dimer levels.