本文關(guān)鍵詞： 富血小板血漿 關(guān)節(jié)軟骨 難愈性創(chuàng)面 修復(fù)　出處：《山東大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：目的：本研究應(yīng)用兩步離心法制備PRP,通過分析PRP中血小板、白細(xì)胞和生長因子的濃度,計(jì)算回收率和富集系數(shù),并做相關(guān)性分析,探討PRP制備方法的實(shí)用性和穩(wěn)定性。關(guān)節(jié)退變臨床產(chǎn)生關(guān)節(jié)疼痛,活動(dòng)受限和關(guān)節(jié)畸形等癥狀,是導(dǎo)致患者長期痛苦和身體殘疾的常見原因,不僅極大地影響了人們的生活質(zhì)量,導(dǎo)致人力資源的喪失,也給家庭和社會(huì)帶來沉重負(fù)擔(dān)。傳統(tǒng)方法存在諸多弊端和生物學(xué)技術(shù)不斷取得新進(jìn)展的今天,PRP技術(shù)為我們治療骨性關(guān)節(jié)炎提供了一種嶄新的治療思路和方法。通過關(guān)節(jié)內(nèi)注射的方法,將PRP與玻璃酸鈉作比較,探討關(guān)節(jié)內(nèi)注射PRP治療關(guān)節(jié)軟骨退行性變的安全性和療效。慢性難愈性創(chuàng)面的治療一直是醫(yī)學(xué)研究的熱點(diǎn),傳統(tǒng)的傷口換藥、清創(chuàng)手術(shù)、負(fù)壓真空吸引和局部應(yīng)用各種藥物等治療,能清除局部感染灶或壞死組織,減少潰瘍分泌物,但仍有許多創(chuàng)面難以愈合,我們采用局部噴灑PRP的方法治療慢性難愈性創(chuàng)面,探索PRP治療慢性難愈性創(chuàng)面的安全性和療效,同時(shí)通過體外實(shí)驗(yàn)評(píng)價(jià)其抗感染作用。方法：(取30例成年健康自愿者捐獻(xiàn)的外周血,男女各15例；年齡24-76歲,平均47歲。用預(yù)先裝有5 mL枸櫞酸鈉注射液的50 mL一次性注射器以18 G針頭于每例志愿者肘前靜脈取血40 mL。搖勻后,取1 mL作血細(xì)胞分析,4 mL作生長因子濃度測定；剩余40 mL注入PRP制備套裝離心管中,于嚴(yán)格無菌狀態(tài)下制備PRP。根據(jù)血液中各組分的沉降系數(shù)不同,以離心半徑15 cm、2000 r/min離心10 min后,全血分為3層,上層為上清液,下層為紅細(xì)胞,兩層交界處一淺黃色層即PRP層。分別將1 mL抗凝靜脈血和制備的PRP采用全自動(dòng)血液分析儀進(jìn)行血常規(guī)分析,測定血小板及白細(xì)胞濃度,并計(jì)算PRP中血小板和白細(xì)胞濃度變異系數(shù)。計(jì)算PRP中血小板或白細(xì)胞回收率和富集系數(shù),回收率=PRP中血小板或白細(xì)胞總數(shù)／全血中血小板或白細(xì)胞總數(shù)×100%,富集系數(shù)=PRP中血小板或白細(xì)胞濃度／全血中血小板或白細(xì)胞濃度。分別測定男、女自愿者PRP血小板及白細(xì)胞濃度。生長因子濃度測定于4 mL抗凝靜脈血中加入0.4 mL凝血酶和氯化鈣混合物(2000 U凝血酶中加入5 mL 5%氯化鈣溶液),3 min后血液凝聚成凍膠狀,常溫下靜置1 h后,同上法離,010 min,吸取上清液轉(zhuǎn)移到凍存管中。將制備的PRP采用相同方法制備得到富含生長因子的上清液。取制備的全血及PRP上清液,按照ELISA試劑盒說明測定PDGF、TGF-β及VEGF濃度。將30例慢性關(guān)節(jié)疼痛或腫脹(不得少于4月),X或MRI有退行性變表現(xiàn)的膝關(guān)節(jié)軟骨退行性變患者納入研究。根據(jù)患者的年齡、性別、體重指數(shù)以及Kellgren Lawrence骨性關(guān)節(jié)炎分級(jí)情況將30例病人分配到PRP組(試驗(yàn)組)和透明質(zhì)酸鈉(Sodium hyaluronate, SH)。試驗(yàn)組15例患者(15膝),每次關(guān)節(jié)內(nèi)注射3.5 mLPRP,對(duì)照組15例患者(15膝),每次關(guān)節(jié)內(nèi)注射透明質(zhì)酸鈉(Sodium hyaluronate, SH) 2 mL。所有患者每3周注射一次,連續(xù)3次為一個(gè)療程。通過記錄治療過程中相關(guān)不良事件的數(shù)量、發(fā)生時(shí)間、嚴(yán)重程度、持續(xù)時(shí)間和處理結(jié)果來評(píng)估該治療方法的安全性。通過比較注射前和3、4、6月隨訪時(shí)的國際膝部文件委員會(huì)(IKDC)主觀膝部評(píng)估表、WOMAC評(píng)價(jià)表、Lequesne指數(shù)評(píng)估表來評(píng)估其臨床療效。將符合選擇標(biāo)準(zhǔn)的21例慢性難愈性創(chuàng)面患者納入本項(xiàng)研究。男8例,女13例；年齡48-78歲,平均58.5歲。開放原始創(chuàng)面,徹底清創(chuàng),按照1：10的比例將制備的2-8mL自體PRP與促凝劑(2000U凝血酶中加入5mL 5%氯化鈣溶液)同時(shí)緩慢噴灑入創(chuàng)面并盡量覆蓋所有創(chuàng)面。3min后創(chuàng)面表面形成一層PRP凝膠,取大小合適的無菌透明薄膜覆蓋于創(chuàng)口,四周密封確保PRP不流失,術(shù)后1周內(nèi)建議患者盡量減少創(chuàng)面臨近關(guān)節(jié)的活動(dòng)量。術(shù)后可視情況更換無菌敷料,一般1周左右查看創(chuàng)面。囑患者避免用手碰觸患處,根據(jù)創(chuàng)面情況決定PRP使用的次數(shù)。采用滴注無菌生理鹽水的方法測定傷口體積和計(jì)算創(chuàng)面覆蓋率,我們將3個(gè)月內(nèi)的創(chuàng)面愈合情況分為如下五個(gè)等級(jí)：Ⅰ級(jí)完全愈合,覆蓋率100%；Ⅱ級(jí)明顯愈合,覆蓋率在75%-100%之間；Ⅲ級(jí)有效愈合,覆蓋率在50%-75%之間；Ⅳ級(jí)部分有效愈合,覆蓋率在25%-50%之間；V級(jí)無效愈合,覆蓋率低于25%。對(duì)于3個(gè)月后沒有痊愈的患者,在征求他們同意的情況下給予再次PRP治療或繼續(xù)觀察治療。于治療前及3月隨訪結(jié)束時(shí)分別行創(chuàng)面內(nèi)分泌物細(xì)菌培養(yǎng),對(duì)比觀察。選取創(chuàng)面感染最常見的金黃色葡萄球菌作為實(shí)驗(yàn)菌株,分別在PRP凝膠及PRP等培養(yǎng)基中孵育,分別于2、4、6、8、12、24h取樣計(jì)數(shù)菌落數(shù),繪制時(shí)間-抑菌曲線圖,計(jì)算細(xì)菌減少率。結(jié)果：研究發(fā)現(xiàn)全血和PRP中血小板濃度分別為(131.40±29.44)×109/L和(819.47±136.32)×109/L,比較差異有統(tǒng)計(jì)學(xué)意義(t=-27.020,P=0.000)；PRP中血小板回收率為60.85%±8.97%,富集系數(shù)為6.40±1.06。全血和PRP中白細(xì)胞濃度分別為(5.57±1.91)×1012/L和(32.20±10.42)×1012/L,比較差異有統(tǒng)計(jì)學(xué)意義(t=-13.780,P=0.000)；PRP中白細(xì)胞回收率為58.30%±19.24%,富集系數(shù)為6.10±1.93。PRP中血小板濃度和白細(xì)胞濃度分別與全血中血小板濃度(r=0.652,P=0.000)和白細(xì)胞濃度(r=0.460,P=0.011)成正相關(guān)。男性組和女性組PRP中血小板濃度和白細(xì)胞濃度比較差異均無統(tǒng)計(jì)學(xué)意義(P0.05)。PRP中PDGF、TGF-β、VEGF濃度分別為(698.15±64.48)、(681.36±65.90)(1071.55±106.04)ng/mL,是全血的(5.67±1.18)、(6.99±0.61)、(5.74±0.83)倍。PRP中PDGF濃度(r=0.832,P=0.020)、TGF-∞濃度(r=0.835,P=0.019)、VEGF濃度(r=0.824,P=0.023)均與PRP中血小板濃度成正相關(guān)。在關(guān)節(jié)軟骨退變部分的臨床研究中未發(fā)生關(guān)節(jié)內(nèi)感染、肌肉萎縮、深靜脈血栓、發(fā)熱、血腫和異常組織增生等不良反應(yīng),與對(duì)照組相比試驗(yàn)組未表現(xiàn)出有更長的不良反應(yīng)持續(xù)時(shí)間(P=0.862),所有出現(xiàn)不良反應(yīng)的患者在得到合理治療后都在4天內(nèi)恢復(fù)到治療前狀態(tài)。試驗(yàn)組患者的評(píng)分在6個(gè)月內(nèi)保持了穩(wěn)定,而對(duì)照組只在前4個(gè)月內(nèi)保持了穩(wěn)定,在第6個(gè)月隨訪時(shí)就表現(xiàn)出明顯的減低(PIDC=0.003, PWOMAC=0.048, PLequesne=0.002)。在前4個(gè)月內(nèi)試驗(yàn)組患者的狀態(tài)改善并不優(yōu)于對(duì)照組(P0.05),但在隨訪到6月時(shí)IKDC評(píng)分和Lequesne指數(shù)的改變提示此時(shí)試驗(yàn)組患者膝關(guān)節(jié)狀態(tài)優(yōu)于對(duì)照組(P,KDC=0.000, PLequesne=0.013)。短期結(jié)果證實(shí)關(guān)節(jié)內(nèi)注射PRP治療關(guān)節(jié)軟骨退行性病變是安全的,其長期療效可能會(huì)優(yōu)于透明質(zhì)酸鈉。在難愈性創(chuàng)面的研究中未發(fā)生創(chuàng)面感染加重、菌血癥、局部異常組織增生等不良反應(yīng)。治療后第1、2、4周隨訪時(shí)創(chuàng)面覆蓋率分別為(55.1±22.6)%、(72.0±16.2)%,(84.3±14.0)%,周間差異有統(tǒng)計(jì)學(xué)意義(P0.05),說明在治療的4周內(nèi),創(chuàng)面處于持續(xù)修復(fù)狀態(tài)。3個(gè)月隨訪時(shí),創(chuàng)面體積為(1.5±1.8)mL,創(chuàng)面平均覆蓋率為85.6%,治愈率為57.1%,治療的有效率和患者主觀滿意率均為95.23%。體外抑菌結(jié)果顯示PRP凝膠抗菌作用明顯,細(xì)菌數(shù)量于孵育4 h時(shí)降至最低。結(jié)論：結(jié)合二次離心法使用PRP制備套裝可以穩(wěn)定地制備出富含高濃度血小板、白細(xì)胞和生長因子的PRP。關(guān)節(jié)內(nèi)注射PRP為臨床治療膝關(guān)節(jié)退行性變提供了一種可能的選擇,但是需要大樣本的隨機(jī)對(duì)照研究的長期隨訪結(jié)果以進(jìn)一步證實(shí)其安全性和療效。PRP不但能有效安全的促進(jìn)慢性難愈性創(chuàng)面的愈合,并有潛在的抗感染作用。意義：通過對(duì)PRP細(xì)胞成分的檢測、抗菌活性的測定等多個(gè)層面的研究,確定了制備PRP的最佳離心時(shí)間及離心力,為PRP在骨科的臨床應(yīng)用提供理論依據(jù)和現(xiàn)實(shí)指導(dǎo)。利用自體PRP來解決關(guān)節(jié)軟骨損傷和難愈性創(chuàng)面修復(fù)的難題,無免疫排斥反應(yīng),制備簡便,費(fèi)用低廉,能有效地縮短治療周期,避免了傳統(tǒng)治療方法效果差及花費(fèi)高等缺點(diǎn),具有良好的社會(huì)和經(jīng)濟(jì)效益,具有廣闊的應(yīng)用前景。
[Abstract]:Objective: To study the two step prepared by centrifugal PRP, through the analysis of PRP in platelet concentration, white blood cells and growth factors, the recovery rate was calculated and the enrichment coefficient, and correlation analysis, to explore the practicability and stability of the preparation methods of PRP. Clinical joint degeneration joint pain, limited mobility and joint deformities and other symptoms that is the result of patients with long-term pain and physical disability common cause, not only greatly affect people's quality of life, resulting in loss of human resources, but also brings heavy burden to family and society. The traditional method has many disadvantages and biological technology have made new progress today, PRP Technology provides a new idea and treatment our method for treatment of osteoarthritis by intra-articular injection. Methods, compare the PRP with sodium hyaluronate intra-articular injection of PRP, to explore the treatment of articular cartilage degeneration safety And the curative effect. For the treatment of chronic refractory wounds is a hotspot of medical research, traditional wound dressing, debridement, negative pressure vacuum and topical application of various drugs and other treatment, can remove local infection or necrosis, reduce ulcer secretions, but there are still many difficult to heal the wound, we adopt the method of local spraying of PRP the treatment of chronic refractory wounds, to explore the safety and efficacy of PRP in treating chronic wounds, and in vitro experiments to evaluate the anti infection effect. Methods: 30 cases of healthy adult volunteers (donor peripheral blood, 15 cases of male and female; the average age is 24-76 years old, 47 years old. The pre loaded with 5 mL citrate acid sodium injection 50 mL disposable syringe with 18 G needle in each volunteers antecubital vein blood 40 mL. after shaking, 1 mL blood cell analysis, 4 mL growth factor concentration determination; the remaining 40 mL injection of PRP preparation Set centrifugal tube, in strict aseptic conditions for preparation of PRP. according to the blood sedimentation coefficient of the components in the different centrifugal radius of 15 cm, 2000 r/min, 10 min after centrifugation, the blood is divided into 3 layers, the upper layer is lower for the supernatant, red blood cell, the two layer at the junction of a light yellow layer is PRP layer respectively. 1 mL anticoagulant venous blood and preparation of PRP using automatic blood analyzer for blood analysis, determination of platelet and leukocyte concentration, PRP in platelet and leukocyte concentration variation coefficient were calculated. The calculation of platelets or white blood cell recovery rate PRP and enrichment coefficient, the recovery rate of platelets or leukocytes in whole blood or / the total number of white blood cells in =PRP * 100%, platelets or leukocytes in whole blood concentration / or leukocyte concentration enrichment coefficient in =PRP were measured. The male, female volunteers PRP platelet and WBC concentration. The concentrations of growth factors measured on 4 mL Add 0.4 mL of thrombin and calcium chloride mixture of anticoagulant venous blood (adding 5 calcium chloride solution for 5% mL 2000 U in 3 min after thrombin), blood condenses into jelly, standing at room temperature for 1 h, 010 min, from the above method, supernatant was transferred to cryotubes. The preparation of PRP use the same method to prepare the supernatant rich in growth factors. Whole blood and PRP supernatant preparation, in accordance with the ELISA kit for determination of PDGF, TGF- and beta VEGF concentration. 30 cases of chronic joint pain or swelling (not less than April), X or MRI showed degeneration of articular cartilage of knee degenerative variable were included in the study. According to the patient's age, gender, body mass index and Kellgren Lawrence osteoarthritis grading of 30 patients were assigned to group PRP (experimental group) and sodium hyaluronate (Sodium hyaluronate, SH). 15 patients in experimental group (15 knees), each intra-articular injection of 3.5 m LPRP, a control group of 15 patients (15 knees), each intra-articular injection of sodium hyaluronate (Sodium hyaluronate SH) 2 mL. all patients were injected once every 3 weeks, 3 times for a course of treatment. The number of records related adverse events during the treatment time of occurrence, severity, duration time and processing results to assess the safety of the treatment. Compared before injection and 3,4,6 month follow-up international documents Committee (IKDC) subjective knee knee scoring scale, WOMAC evaluation, Lequesne index evaluation table to evaluate the clinical curative effect. The study included 21 cases of chronic wound patients will meet the selection criteria. 8 cases were male, 13 were female; the average age is 48-78 years old, 58.5 years old. The open wound debridement, in accordance with the 1:10 proportion will be prepared 2-8mL autologous PRP with coagulant (adding calcium chloride solution for 5% 5mL 2000U in thrombin) at the same time slowly into a spray And try to cover all the wounds after.3min wound surface to form a layer of PRP gel, taking the sterile transparent film of suitable size to cover the wound, to ensure no loss of seal around PRP, suggesting that patients should reduce the wound near the joint activity within 1 weeks after surgery. After surgery can replace a sterile dressing, generally about 1 weeks to see the wound avoid touching the affected area. The patients with hand wound, number according to the decision PRP use. Determination of wound volume and calculate the wound coverage by infusion of sterile saline, we will be within 3 months of the wound healing is divided into the following five grades: Grade I completely healed. The coverage rate of 100%; grade II significantly healing coverage between 75%-100%; III effective healing, coverage of 50%-75%; Part IV effective healing, coverage of 25%-50%; V invalid healing, coverage is lower than 25%. for 3 months After not healed patients treated observation PRP treatment again or continue to seek their consent. At the end of observation before treatment and at follow-up in March were cultured within the wound. The wound secretions of bacteria selected the most common infection of Staphylococcus aureus as the experimental strains were incubated in medium PRP gel PRP and 2,4,6,8,12,24h, respectively in the sample count the number of colonies, rendering time antibacterial curve, calculation bacteria reduction rate. Results: the study found that PRP in whole blood and platelet concentrations were (131.40 + 29.44) * 109/L and (819.47 + 136.32) * 109/L, the difference was statistically significant (t=-27.020, P=0.000); for 60.85% + 8.97% recovery rate of platelet PRP, white blood cell concentration enrichment factor was 6.40 + 1.06. blood and PRP were (5.57 + 1.91) * 1012/L and (32.20 + 10.42) * 1012/L, the difference was statistically significant (t=-13 .780, P=0.000); 58.30% + 19.24% white blood cell recovery rate PRP, enrichment coefficient was 6.10 + 1.93.PRP in platelet and leukocyte concentration and concentration respectively in whole blood platelet concentration (r=0.652, P=0.000) and leukocyte concentration (r=0.460, P=0.011) were positively correlated. Platelet concentration and leukocyte concentration difference between male group and women in the PRP group had no statistical significance (P0.05) in.PRP PDGF, TGF- beta, VEGF concentrations were (698.15 + 64.48), (681.36 + 65.90) (1071.55 + 106.04) ng/mL, the blood is (5.67 + 1.18), (6.99 + 0.61), (5.74 + 0.83) PDGF concentration times.PRP (r=0.832, P=0.020), TGF- (r=0.835, P=0.019) - concentration, the concentration of VEGF (r=0.824, P=0.023) were positively correlated with PRP concentration in platelet. No joint infection, muscle atrophy and degeneration of articular cartilage in the clinical research part, deep vein thrombosis, fever, adverse reactions and abnormal tissue hematoma hyperplasia, Compared with the control group, experimental group showed no adverse reaction with longer duration (P=0.862), all patients with adverse reactions to recover before treatment in a reasonable state after treatment in 4 days. In the test group the score remained stable in 6 months, while the control group only in the first 4 months remained stable, in sixth months follow-up showed significantly decreased (PIDC=0.003, PWOMAC=0.048, PLequesne=0.002). In the first 4 months of test group improvement was better than the control group (P0.05), but in the follow-up to June when the IKDC score and Lequesne index change it in the test group the knee joint state is better than the control group (P, KDC=0.000, PLequesne=0.013). The short-term results confirmed that intra-articular injection of PRP for the treatment of articular cartilage degeneration is safe, may the long-term efficacy is superior to sodium hyaluronate. In the study of more difficult wounds No wound infection occurred aggravating, bacteremia, adverse reactions of local abnormal tissue hyperplasia. Treatment after 1,2,4 weeks wound coverage rates were (55.1 + 22.6)% and (72 + 16.2)% and (84.3 + 14)%, there was statistical significance difference between the week (P0.05), in the treatment of within 4 weeks, continue to repair wounds in a state of.3 months of follow-up, the wound volume (1.5 + 1.8) mL, the average wound coverage rate was 85.6%, the cure rate was 57.1%, the treatment efficiency and subjective satisfaction rate of patients was 95.23%. in vitro showed significant antibacterial effect of PRP gel, the number of bacteria in the at 4 h of incubation to a minimum. Conclusion: the combination of two centrifugal method using PRP preparation kit can be stably prepared with high concentration of platelets, white blood cells and growth factor PRP. intra-articular injection of PRP provides a possible choice for the clinical treatment of knee joint degeneration, but requires a large sample Long term follow-up results of randomized studies to further confirm the safety and efficacy of.PRP security can not only effectively promote the healing of chronic wounds, and anti infection effect. The potential significance: through the detection of PRP cell components, research activity determination and many other aspects of antibacterial, the optimum centrifugal time the centrifugal force and the preparation of PRP PRP, to provide a theoretical basis and practical guidance in the clinical application of Department of orthopedics. Using autologous PRP to solve the problem of repairing articular cartilage injury and wound healing, no immune rejection, simple preparation, low cost, can effectively shorten the treatment cycle, to avoid the effect of traditional treatment method and the cost higher shortcomings, it has good social and economic benefits, and has broad application prospects.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R684

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