PLGA静电纺丝膜对术后腹膜粘连预防效果研究
摘要
PLGA静电纺丝膜，集合了静电纺丝和高分子聚合物两者的优点，不仅具有高孔隙率，高透氧率，同时还具有良好的生物相容性及很好的成膜性，并能在体内自动降解。它的孔结构不仅可以使伤口的液体及时渗出同时又能保证伤口保持一定的湿润性，还能防止细菌的入侵，给组织生长创造了良好的环境，在组织愈合后，它又能自动降解，不会在体内留下任何异物，也不会给机体带来任何的毒副作用，以上所述优点，都为其能成为良好的防粘连隔离物奠定了基础。本文主要对PLGA静电纺丝膜的生物相容性及防粘连效果进行研究，并进行了大鼠体内试验，其主要内容及结果如下：
1. PLGA静电纺丝膜的体外细胞试验
（1）将PLGA静电纺丝膜和IEC-6、NIH3T3两种细胞系进行共培养，并进行CCK-8检测，结果显示PLGA静电纺丝膜无细胞毒性；
（2）将IEC-6、NIH3T3两种细胞系培养在不同PLGA静电纺丝膜上，然后经过一系列处理，经MTT法测定，与对照组相比，结果显示细胞不易粘附在PLGA静电纺丝膜上；
（3）对孵育在不同的膜上的IEC-6、NIH3T3两种细胞系进行荧光染色、拍照，观察可知，粘附在PLGA膜上细胞要比对照组的少；
（4）将PLGA静电纺丝膜降解液与IEC-6、NIH3T3两种细胞系共培养，并进行相应处理，然后用扫描电镜观察其微观形态，表明处理组的细胞形态与对照组无明显变化。
2. PLGA静电纺丝膜防粘连体内试验
（1）通过建立动物模型，进行动物试验，观察处死后大鼠的并发症及粘连情况，结果表明使用了PLGA静电纺丝膜作为隔离物的动物模型相对于对照组，其并发症发病几率明显降低，粘连几率也显著降低；
（2）将不同时期不同组别的大鼠血液进行检测，其红细胞计数、白细胞计数都没有明显变化，说明PLGA静电纺丝膜无明显免疫原性，不易引起大鼠过敏。
（3）对处死前的大鼠血液进行凝血酶原时间、活化部分凝血活化酶时间、凝血酶凝固时间检测，所有的动物的以上三个指标均在正常范围内，没有出现凝血不正常的现象，说明PLGA静电纺丝膜对大鼠的凝血功能没有影响;
（4）急性期大鼠腹腔灌洗液SOD值比较：阴性组（未做相应隔离处理）SOD值明显低于正常组。其它无显著变化。
亚急性期大鼠腹腔灌洗液SOD值比较：阴性组（未做相应隔离处理）、阳性组（以商业膜为隔离物）SOD都显著低于正常组（P<0.05）。其它无显著变化。
慢性期大鼠腹腔灌洗液SOD值比较：阴性组（未做相应隔离处理）极显著低于正常组（P<0.01），阳性组（以商业膜为隔离物）显著低于正常组（P<0.05）。
（5）急性期大鼠腹腔灌洗液MDA值比较：阴性组（未做相应隔离处理）和阳性组（以商业膜为隔离物）MDA值极显著高于正常组（P<0.01）。
亚急性期大鼠腹腔灌洗液MDA值比较：阴性组（未做相应隔离处理）MDA值显著高于正常组（P<0.05）。其它组无显著变化。
慢性期大鼠腹腔灌洗液MDA值比较：阴性组（未做相应隔离处理）MDA值显著高正常组（P<0.05）。
3.结论
体外细胞试验结果表明，PLGA静电纺丝膜无细胞毒性，并能有效防止细胞在膜上粘附。通过大鼠体内试验，观察并发症及粘连发生情况，结果显示，PLGA静电纺丝膜可以明显降低并发症及粘连发生的几率。大鼠血液血常规及凝血指标检测结果显示， PLGA静电纺丝膜无明显免疫原性，对大鼠的凝血功能无影响。通过测定大鼠急性期、亚急性期、慢性期腹腔灌洗液SOD值和MDA值，并做比较，得出， PLGA静电纺丝膜有助于大鼠自身自由基清除能力的恢复，显著改善局部粘膜损害，加速伤口的愈合。
PLGA electrostatic spinning film, combined with advantages of electrospinning and highmolecular polymer, not only have high porosity and high-oxygen transmission rate, but alsohave good biocompatibility，good film-forming ability and autoproteolysis. Its propertiesallow the wound liquid oozing in time, and ensure that the wound wettability. It can protecttissue against the bacteria, and then create a favorable environment for tissue growth andhealing. It can automatically degrade without leaving foreign matter and toxic andside-effects.
This paper focused on the study of the toxicity of PLGA electrostatic spinning film,effects of anti-adhesion, and rats’ in vivo tests, and the results are showed as follows:
1．The cell test of PLGA membrane
(1) Through co-culture the PLGA membrane with IEC-6and NIH3T3, detected byCCK-8test, the results show that the PLGA membrane is not of cell toxicity.
(2) In the MTT assay, two of cell lines IEC-6, NIH3T3were incubated in the differentPLGA film by a series of treatments against the control, the PLGA film does not adhered bythe cell easily.
(3) The two of cell lines IEC-6, NIH3T3were incubated at different membranes andtaking pictures assisted by the fluorescent dye. It could be observed that thenumber of thecells adhered to the PLGA membranes were less than that of the control group.
(4) Co-cultured in the PLGA membrane degradation solution with IEC-6and NIH3T3cells, respectively, the morphology observed by scanning electron microscope shows thatthere is no significant difference between treatment and control.
2．In vivo test of PLGA membrane anti-adhesion
(1) The animal models were established. Complications and adhesions were observed.The results showed that the PLGA electrospun membrane significantly reduced the risk ofcomplications and adhesion compared with the control.
(2) There is not significantly difference between the number of the red blood cell andwhite blood cell, indicating that the PLGA membrane has no apparent toxicity and irritation.
(3) Analyzing the prothrombin time, the activated partial thromboplastin time and theprothrombin coagulating time, all of these three indicators are within the normal range. Thereis no coagulating phenomenon. It shows that the PLGA membrane does not affect thecoagulation of the blood in rats.
(4) During the acute phase, the SOD value of the negative group was significantly lower than the normal group. The SOD value of the160μm of the PLGA membrane group wassignificantly higher than that of the negative group (P <0.05). The others had no significantchange.
The SOD of Sub-acute rat of peritoneal lavage fluid: the SOD value of negative group,positive group, PLGA membrane group, the SOD value of the60μm group were significantlylower than that of the normal group (P <0.05). The others had no significant difference.
The SOD of chronic rat of peritoneal lavage fluid: the SOD value of the negative groupwas significantly lower than that of the normal group (P <0.01). The SOD value of thepositive was significantly lower than that of the normal group (P <0.05). The SOD value ofthe positive group and the160μm PLGA membrane group were significantly higher than thatof negative group (P <0.01).
(5) The MDA of the acute phase rat of peritoneal lavage fluid: the MDA of the negativegroup and positive group were significantly higher than that of the normal group (P <0.01).The MDA values of the160μm of PLGA film and the60μm PLGA membrane group weresignificantly higher than that of the normal group (P <0.05).
The MDA of sub-acute rat peritoneal lavage fluid: the MDA of the negative group wassignificantly higher than that of the normal group (P <0.05). Other groups have no significantchanges.
The MDA of Chronic rat peritoneal lavage fluid: The MDA of negative group issignificantly higher than that of normal group (P <0.05). The MDA of the positive group andthe160μm PLGA membrane group is significantly lower than that of the negative group (P<0.01).
3. Conclusion
The results of the PLGA electrostatic spinning film show that，it has no cytotoxicity, andcan effectively prevent the cell adhering on the membrane. By the in vivo test, thecomplications and adhesion can significantly reduce by applying the PLGA electrostaticspinning films. The results of routine blood and coagulation index test of rats show that thereis no immunogenicity and no effect on rats’ blood coagulation. Compared with the SOD andthe MDA values of acute phase, subacute, and chronic phase of peritoneal lavage fluid of rats,the PLGA electrostatic spinning film contribute to the capacity of rat's own free radicalscavenging recovery, significant improve mucosal damage, speed up wound healing.
1. PLGA静电纺丝膜的体外细胞试验
（1）将PLGA静电纺丝膜和IEC-6、NIH3T3两种细胞系进行共培养，并进行CCK-8检测，结果显示PLGA静电纺丝膜无细胞毒性；
（2）将IEC-6、NIH3T3两种细胞系培养在不同PLGA静电纺丝膜上，然后经过一系列处理，经MTT法测定，与对照组相比，结果显示细胞不易粘附在PLGA静电纺丝膜上；
（3）对孵育在不同的膜上的IEC-6、NIH3T3两种细胞系进行荧光染色、拍照，观察可知，粘附在PLGA膜上细胞要比对照组的少；
（4）将PLGA静电纺丝膜降解液与IEC-6、NIH3T3两种细胞系共培养，并进行相应处理，然后用扫描电镜观察其微观形态，表明处理组的细胞形态与对照组无明显变化。
2. PLGA静电纺丝膜防粘连体内试验
（1）通过建立动物模型，进行动物试验，观察处死后大鼠的并发症及粘连情况，结果表明使用了PLGA静电纺丝膜作为隔离物的动物模型相对于对照组，其并发症发病几率明显降低，粘连几率也显著降低；
（2）将不同时期不同组别的大鼠血液进行检测，其红细胞计数、白细胞计数都没有明显变化，说明PLGA静电纺丝膜无明显免疫原性，不易引起大鼠过敏。
（3）对处死前的大鼠血液进行凝血酶原时间、活化部分凝血活化酶时间、凝血酶凝固时间检测，所有的动物的以上三个指标均在正常范围内，没有出现凝血不正常的现象，说明PLGA静电纺丝膜对大鼠的凝血功能没有影响;
（4）急性期大鼠腹腔灌洗液SOD值比较：阴性组（未做相应隔离处理）SOD值明显低于正常组。其它无显著变化。
亚急性期大鼠腹腔灌洗液SOD值比较：阴性组（未做相应隔离处理）、阳性组（以商业膜为隔离物）SOD都显著低于正常组（P<0.05）。其它无显著变化。
慢性期大鼠腹腔灌洗液SOD值比较：阴性组（未做相应隔离处理）极显著低于正常组（P<0.01），阳性组（以商业膜为隔离物）显著低于正常组（P<0.05）。
（5）急性期大鼠腹腔灌洗液MDA值比较：阴性组（未做相应隔离处理）和阳性组（以商业膜为隔离物）MDA值极显著高于正常组（P<0.01）。
亚急性期大鼠腹腔灌洗液MDA值比较：阴性组（未做相应隔离处理）MDA值显著高于正常组（P<0.05）。其它组无显著变化。
慢性期大鼠腹腔灌洗液MDA值比较：阴性组（未做相应隔离处理）MDA值显著高正常组（P<0.05）。
3.结论
体外细胞试验结果表明，PLGA静电纺丝膜无细胞毒性，并能有效防止细胞在膜上粘附。通过大鼠体内试验，观察并发症及粘连发生情况，结果显示，PLGA静电纺丝膜可以明显降低并发症及粘连发生的几率。大鼠血液血常规及凝血指标检测结果显示， PLGA静电纺丝膜无明显免疫原性，对大鼠的凝血功能无影响。通过测定大鼠急性期、亚急性期、慢性期腹腔灌洗液SOD值和MDA值，并做比较，得出， PLGA静电纺丝膜有助于大鼠自身自由基清除能力的恢复，显著改善局部粘膜损害，加速伤口的愈合。
PLGA electrostatic spinning film, combined with advantages of electrospinning and highmolecular polymer, not only have high porosity and high-oxygen transmission rate, but alsohave good biocompatibility，good film-forming ability and autoproteolysis. Its propertiesallow the wound liquid oozing in time, and ensure that the wound wettability. It can protecttissue against the bacteria, and then create a favorable environment for tissue growth andhealing. It can automatically degrade without leaving foreign matter and toxic andside-effects.
This paper focused on the study of the toxicity of PLGA electrostatic spinning film,effects of anti-adhesion, and rats’ in vivo tests, and the results are showed as follows:
1．The cell test of PLGA membrane
(1) Through co-culture the PLGA membrane with IEC-6and NIH3T3, detected byCCK-8test, the results show that the PLGA membrane is not of cell toxicity.
(2) In the MTT assay, two of cell lines IEC-6, NIH3T3were incubated in the differentPLGA film by a series of treatments against the control, the PLGA film does not adhered bythe cell easily.
(3) The two of cell lines IEC-6, NIH3T3were incubated at different membranes andtaking pictures assisted by the fluorescent dye. It could be observed that thenumber of thecells adhered to the PLGA membranes were less than that of the control group.
(4) Co-cultured in the PLGA membrane degradation solution with IEC-6and NIH3T3cells, respectively, the morphology observed by scanning electron microscope shows thatthere is no significant difference between treatment and control.
2．In vivo test of PLGA membrane anti-adhesion
(1) The animal models were established. Complications and adhesions were observed.The results showed that the PLGA electrospun membrane significantly reduced the risk ofcomplications and adhesion compared with the control.
(2) There is not significantly difference between the number of the red blood cell andwhite blood cell, indicating that the PLGA membrane has no apparent toxicity and irritation.
(3) Analyzing the prothrombin time, the activated partial thromboplastin time and theprothrombin coagulating time, all of these three indicators are within the normal range. Thereis no coagulating phenomenon. It shows that the PLGA membrane does not affect thecoagulation of the blood in rats.
(4) During the acute phase, the SOD value of the negative group was significantly lower than the normal group. The SOD value of the160μm of the PLGA membrane group wassignificantly higher than that of the negative group (P <0.05). The others had no significantchange.
The SOD of Sub-acute rat of peritoneal lavage fluid: the SOD value of negative group,positive group, PLGA membrane group, the SOD value of the60μm group were significantlylower than that of the normal group (P <0.05). The others had no significant difference.
The SOD of chronic rat of peritoneal lavage fluid: the SOD value of the negative groupwas significantly lower than that of the normal group (P <0.01). The SOD value of thepositive was significantly lower than that of the normal group (P <0.05). The SOD value ofthe positive group and the160μm PLGA membrane group were significantly higher than thatof negative group (P <0.01).
(5) The MDA of the acute phase rat of peritoneal lavage fluid: the MDA of the negativegroup and positive group were significantly higher than that of the normal group (P <0.01).The MDA values of the160μm of PLGA film and the60μm PLGA membrane group weresignificantly higher than that of the normal group (P <0.05).
The MDA of sub-acute rat peritoneal lavage fluid: the MDA of the negative group wassignificantly higher than that of the normal group (P <0.05). Other groups have no significantchanges.
The MDA of Chronic rat peritoneal lavage fluid: The MDA of negative group issignificantly higher than that of normal group (P <0.05). The MDA of the positive group andthe160μm PLGA membrane group is significantly lower than that of the negative group (P<0.01).
3. Conclusion
The results of the PLGA electrostatic spinning film show that，it has no cytotoxicity, andcan effectively prevent the cell adhering on the membrane. By the in vivo test, thecomplications and adhesion can significantly reduce by applying the PLGA electrostaticspinning films. The results of routine blood and coagulation index test of rats show that thereis no immunogenicity and no effect on rats’ blood coagulation. Compared with the SOD andthe MDA values of acute phase, subacute, and chronic phase of peritoneal lavage fluid of rats,the PLGA electrostatic spinning film contribute to the capacity of rat's own free radicalscavenging recovery, significant improve mucosal damage, speed up wound healing.
引文
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