富血小板血浆对大鼠背部超长随意皮瓣成活的影响

Effects of platelet-rich plasma on the survival of ultra-long dorsal random flaps in rats

目的观察富血小板血浆(PRP)对大鼠背部超长随意皮瓣成活的影响。方法取16只鼠龄6~8周雄性SD大鼠(下同)全血,每只9~10mL,采用改良APPLE法制备PRP;全自动血液细胞分析仪对全血及PRP进行血小板计数,检测PRP制作成功。另取32只大鼠,按随机数字表法分为PRP组和对照组,每组16只。于每只大鼠背部制成1个矩形超长随意皮瓣并原位回植,皮瓣面积为8cm×2cm。在皮瓣两侧分别设计等距的3点,PRP组大鼠从每个注射点真皮及皮下注射0.1mLPRP,对照组大鼠注射同等体积的生理盐水。术后7d,观察大鼠皮瓣成活情况并计算皮瓣成活率。术后7d,每组处死8只大鼠,取皮瓣近、中、远端组织,苏木精-伊红染色观察皮瓣组织学变化。取术后24h(每组处死8只大鼠)、7d2组大鼠距蒂部3~4cm处皮瓣组织,实时荧光定量反转录PCR检测血管内皮生长因子(VEGF)、血小板源性生长因子AA(PDGF-AA)和PDGF-BBmRNA表达量,硝酸还原酶法测定一氧化氮含量。对数据行t检验。结果(1)术后7d,PRP组大鼠皮瓣较干燥,未出现脓性分泌物,皮瓣远端出现痂壳附着,痂壳自然脱落后露出色泽淡红愈合皮肤;对照大鼠皮瓣可见大量炎性渗出物,皮瓣远端1/2~2/3发生坏死且形成痂壳,痂壳不易剥脱。PRP组大鼠皮瓣成活率为(67±6)%,明显高于对照组的(52±10)%,t=1.94,P<0.05。(2)PRP组大鼠近端皮瓣未见明显炎性细胞浸润,纤维组织排列整齐,较多微血管形成;中端皮瓣可见少量炎性细胞浸润,纤维组织排列稍紊乱,较多微血管形成。远端皮瓣纤维组织排列较混乱,较多炎性细胞浸润,较多微血管形成,有少量血管栓塞。对照组大鼠近、中、远端皮瓣均有大量炎性细胞浸润,纤维组织排列混乱,微血管形成较少。(3)术后24h、7d,PRP组大鼠VEGF、PDGF-AA及PDGF-BBmRNA的表达量明显高于对照组(t=6.46、5.61、2.88、10.18、6.10、7.67,P<0.001)。(4)术后24h,PRP组大鼠皮瓣组织一氧化氮含量为(5.0±0.9)μmol/g,明显高于对照组的(3.4±0.9)μmol/g(t=19.14,P<0.001)。术后7d,PRP组大鼠皮瓣组织一氧化氮含量为(3.3±0.8)μmol/g,与对照组大鼠的(3.0±0.6)μmol/g相近(t=2.93,P>0.05)。结论PRP能改善大鼠背部超长随意皮瓣的成活率,可能与PRP可通过调节血管生成相关因子,提高一氧化氮含量,抑制细胞过度凋亡,从而减轻缺血再灌注损伤,改善微循环障碍有关。

Objective To observe the effects of platelet-rich plasma (PRP) on the survival of ultra-long dorsal random flaps in rats. Methods Sixteen male Sprague Dawley rats aged 6 to 8 weeks (the same below) were sacrificed to collect whole blood of 9 to 10 mL from each rat, and PRP was prepared by modified APPLE method. The platelet count of retained whole blood and PRP detected by automated blood cell analyzer showed that PRP was made successfully. The other thirty-two rats were collected and divided into PRP group and control group according to the random number table, with 16 rats in each group. One rectangular ultra-long random flap with area of 8 cm×2 cm was made on the back of each rat and replanted in situ. The equidistant 3 points were designed on both sides of the flap of each rat. Rats in PRP group were injected with 0.1 mL PRP from dermis and subcutaneous tissue of each injection point, while rats in control group were injected with the same volume of normal saline. Eight rats in each group were sacrificed at post operation hour (POH) 24 and on post operation day (POD) 7. On POD 7, survival of flaps of rats in 2 groups was observed, and the survival rates of flaps were calculated. On POD 7, the proximal, middle, and distal flaps of rats in 2 groups were collected, and histological changes of the flaps of rats in 2 groups were observed with hematoxylin-eosin staining. At POH 24 and on POD 7, flaps in 3 to 4 cm to pedicles were taken to detect mRNA expressions of vascular endothelial growth factor (VEGF), platelet-derived growth factor AA (PDGF-AA) and PDGF-BB by real-time fluorescent quantitative reverse transcription polymerase chain reaction, and to determine content of nitric oxide by nitrate reductase method. Data were processed with t test. Results (1) On POD 7, flaps of rats in PRP group were dry without purulent exudate, and covered with scab, and the pink new skin emerged after scab fell off. On POD 7, flaps of rats in control group were with a large amount of inflammatory exudates, 1/2 to 2/3 of flaps at the distal were with necrosis and covered by scab which was not easy to be stripped. The survival rate of flap of rats in PRP group was (67±6)%, significantly higher than (52±10)% of rats in control group (t=1.94, P<0.05). (2) There were no obvious inflammatory cell infiltration and a number of microvessels, and fibrous tissue arranged neatly in the proximal flaps of rats in PRP group. There were a few of inflammatory cell infiltration and a number of microvessels, and fibrous tissue arranged slightly disorderly in the middle flaps of rats in PRP group. There were many more inflammatory cell infiltration and microvessels, a small amount of vascular embolism, and fibrous tissue arranged disorderly in the distal flaps of rats in PRP group. There were a large number of inflammatory cells infiltration and a few of microvessels, and fibrous tissue arranged disorderly in the proximal, middle, and distal flaps of rats in control group. (3) At POH 24 and on POD 7, mRNA expressions of VEGF, PDGF-AA, and PDGF-BB of rats in PRP group were significantly higher than those of rats in control group (t=6.46, 5.61, 2.88, 10.18, 6.10, 7.67, P<0.001). (4) At POH 24, content of nitric oxide in flap of rats in PRP group was (5.0±0.9) μmol/g, significantly higher than (3.4±0.9) μmol/g of rats in control group (t=19.14, P<0.001). On POD 7, content of nitric oxide in flap of rats in PRP group was (3.3±0.8) μmol/g, which was close to (3.0±0.6) μmol/g of rats in control group (t=2.93, P>0.05). Conclusions PRP can improve the survival rate of ultra-long dorsal random flap in rats, which may be related to regulation of angiogenesis related factors, increase of nitric oxide content, and inhibition of excessive apoptosis of cells of PRP, so as to alleviate ischemical reperfusion injury and improve microcirculatory disturbance.