壳聚糖止血活性及其生物安全性评价研究
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摘要
壳聚糖是一种具有止血促愈活性的生物可降解的天然多糖，近年来已成为止血性生物材料的研究热点之一，目前已有多种外用壳聚糖基敷料获批上市。但受限于其在体内的止血效果和生物安全性尚不明确，壳聚糖体内止血材料的开发和应用仍处于起步阶段。本论文旨在在动物体内对壳聚糖的止血能力和生物安全性进行深入系统的评价，为壳聚糖基体内植入型止血敷料的开发奠定研究基础，为其应用提供理论依据，对壳聚糖产业的发展具有重要的理论价值和应用价值。
为了筛选出具有良好止血活性的壳聚糖并评价其在体内的止血效果，利用试管法对比研究一系列具有不同脱乙酰度（DD）、分子量（Mw）的壳聚糖固体粉末和溶液在体外对兔血凝固时间的影响，利用小鼠断尾法测定不同性质壳聚糖粉末对鼠尾出血时间和出血量的影响，并将据此筛选得的壳聚糖样品应用于兔体内脏器的损伤创口，测定各创口的出血时间和出血量的变化。结果显示：壳聚糖样品的Mw和DD对其体外止血活性影响显著，Mw和DD越高，兔血凝固时间越短，且同等剂量下壳聚糖粉末的促凝血活性优于其溶液；将各壳聚糖粉末样品应用于小鼠断尾创口，出血时间均可缩短53.4%以上，DD为85.32%、Mw为1.22×106的壳聚糖粉末（CS）更可将出血时间缩短74.7%，并将出血量减少86.4%；将该CS粉末应用于动物体内脏器创口，最高可缩短出血时间63.5%、减少出血量72.9%，对体内的多血管实质性脏器（如肝、脾）的出血创口具有显著的止血效果。
为了探讨CS发挥体内止血活性的可能途径，将其与广泛分布于血管内腔表面的内皮细胞共培养24h，对于细胞合成与分泌的促凝血因子血管性假性血友病因子（vWF）、组织因子（TF）、纤溶酶原活化物抑制剂（PAI）、内皮素（ET-1）和抗凝血因子内皮型一氧化氮合成酶（ENOs）、血栓调节蛋白（TM）、蛋白S（PS）、组织型纤溶酶原活化物（tPA）、组织因子途径抑制因子（TFPI）等mRNA表达量的变化，用RT-PCR和荧光定量分析法进行测定。结果表明：CS对抗凝血因子TFPI、tPA和ENOs的mRNA表达均有一定的抑制作用，高剂量CS（1mg/mL）更可使其表达量分别降低至空白对照组的34.40%、0.77%和9.24%。因此，通过抑制内皮细胞抗凝血因子的合成分泌而使血液加速凝固，可能是CS体内止血的途径之一。
对于体内止血效果显著的CS的生物安全性，重点从以下几方面进行了研究。
(1)急性毒性和长期毒性：分别按照2000mg/kg和5000mg/kg的限度剂量于ICR小鼠腹腔注射CS混悬液，观察短期内动物的生存状况和不良反应症状，检查其血液学和组织学变化，以此评价其急性毒性；对小鼠腹腔注射CS混悬液后持续观察90d，根据动物的生存状况、不良反应、血液学和组织学检查结果评价其长期毒性。结果显示：腹腔注射CS混悬液的LD_(50)>5000mg/kg，属于低毒物质；小鼠出现体重和摄食量显著下降、毛发竖立、腹泻、活动量急剧减少等短期临床毒性反应，机体产生对外来物质的急性炎症反应，肝和肾有显著的炎症反应，脾脏因其白髓弥散性增生而显著增大；CS在小鼠体内的长期存在，可引起机体慢性炎症、脾脏持续增大和血小板数量持续增加。
(2)遗传毒性和细胞毒性：通过Ames实验、小鼠微核实验和精子畸形实验对CS的遗传毒性和致突变作用进行了研究，同时利用MTT法研究了CS对内皮细胞HUVEC增殖率的影响，以此表征其细胞毒性。结果显示：在实验剂量范围内，CS未引起测试菌株或细胞的异常变化，无显著的遗传毒性；CS可促进HUVEC增殖，在0.0625~1mg/mL范围内无细胞毒性。
(3)生殖毒性、发育毒性和致畸毒性：小鼠分别于交配前和妊娠早期（GD6）腹腔注射CS，观察亲代F0小鼠的生殖参数和交配、受精、妊娠、分娩及哺乳等过程的变化，记录其胚胎形成、发育及子代的生长和畸形状况。结果显示：CS对F0雄鼠的生殖参数未有显著影响，但低剂量CS（125mg/kg）即可引起F0雌鼠黄体数和着床数的显著下降，从而导致活胎数的减少，因此，CS对小鼠生殖毒性的无明显损害作用水平（NOAEL）低于125mg/kg；中剂量CS（500mg/kg）即可引起F0孕鼠消瘦和先兆流产，其活胎数显著减少，但其仔鼠的生长发育未受显著影响，因此，CS对小鼠发育毒性的NOAEL为125mg/kg；同时，各剂量组小鼠畸形率无显著差异，CS的致畸毒性NOAEL大于2000mg/kg。
综上所述，根据体外和动物体表止血实验筛选得DD为85.32%、Mw为1.22×106的壳聚糖样品CS，其应用于动物体内脏器创口时，可显著缩短出血时间并降低出血量，具有良好的体内止血效果，具有较好的开发和研究前景；首次对作为体内止血材料的壳聚糖样品进行系统的生物安全性评价，结果显示其在小鼠体内的LD_(50)>5000mg/kg，无遗传毒性、细胞毒性和致畸毒性，但是亦可引起一定的临床不良反应，尤其是对雌鼠的生殖和孕鼠的妊娠过程影响较大，该结果对于壳聚糖体内止血敷料的开发和应用具有重要的理论价值和指导意义。
Chitosan is a natural biodegradability polysaccharide with hemostasis and wound healingactivities. It has been a research highlight as a kind of hemostatic biomaterials in recent years,and there are several kinds of topical chitosan-based dressing approved for listing. However,the development and application of chitosan as an implanted hemostatic material are still intheir infancy, which is limited by its unclear hemostatic effect and biological safety in vivo.The purposes of this thesis are to evaluate the hemostatic capacity and biological safety ofchitosan in animals, to lay research foundations for its development and offer academic basesfor its application as an in vivo hemostatic dressing, and this has important theoretical andpractical values in the developments of chitosan industry.
In order to screen a sample with good hemostatic activity and evaluate its hemostaticcapability in vivo, the effects of a series of chitosan powder and solution with different DDand Mw to the coagulation time of rabbit blood in vitro were studied by Lee-White method,and the effects of chitosan powder samples with different properties to the time and theamount of bleeding of mouse tail were investigated by tail cutting method. The samplescreened according to these was applied to the organ wounds of rabbits in vivo, and the timeand amount of bleeding were measured. The results showed that the Mw and DD had greateffects on the hemostatic activity of chitosan, the coagulation time was shorter while the Mwand DD were higher, and the activity of powder was better than the solution at the same dose.All chitosan powder samples could shorten the bleeding time of mouse tail more than53.4%and CS powder, whose DD was85.32%and Mw was1.22×106, could reduce the bleedingtime by74.7%and the amount of bleeding by86.4%. CS powder showed a good hemostaticeffect on the bleeding wounds of substantive organs with rich blood (such as liver and spleen),while the bleeding time could be shortened63.5%and the amount reduced72.9%at most.
To explore the possible way of CS to play its hemostatic activity in vivo, it wasco-cultured with endothelial cells, which were widely distributed in the vessel lumen surface,for24h. The changes of procoagulant factors (such as vWF, TF, PAI, and ET-1) andanti-coagulant factors (such as ENOs, TM, PS, tPA, and TFPI) synthesized and secreted byendothelial cells were measured by RT-PCR and fluorescence quantitative analysis, whichcould denote the changes of coagulation capability of endothelial cells. The results showedthat CS could inhibited the mRNA expressions of TFPI, tPA, and ENOs, and their expressionamounts in the highest CS group were34.40%,0.77%, and9.24%of control group,respectively. Therefore, CS could promote blood clotting by inhibiting the synthesis andsecretion of anti-coagulant factors, and this might be one way of chitosan to stop the bleedingin vivo.
The key researches about the biological safety of CS were as follow.
(1) Acute and chronic toxicity: CS suspension was intraperitoneally injected into ICRmice at2000mg/kg and5000mg/kg, respectively. The survival condition and symptoms ofadverse effects were observed for14days, and hematological and histological tests wereperformed. Then CS suspension was singly injected intraperitoneally into ICR mice and the observation lasted for90days. The survival condition, symptoms of adverse effects,hematological and histological changes were observed to evaluate the chronic toxicity. Theresults showed that the LD_(50)of CS was over5000mg/kg in mice, and it was a less-toxicmaterial. Some adverse effects, such as decreasing of body weight and food consumption,erected hair, serious diarrhea, and sharply reduced movements, were observed. Acuteinflammations of body to strange materials were noted, inflammatory cells were increased inlivers and kidneys, and spleens were enlarged notably for the disseminated hyperplasis ofwhite pulp area. The long-term existence of CS in vivo could induce chronic inflammation ofbody, persistive enlargement of spleen, and continued increasement of platelet.
(2) Genotoxicity and cytotoxicity: the genotoxicity and mutagenicity of CS were studiedby Ames test, mouse micronucleus test and sperm abnormality test, and the effect of CS to theproliferation of HUVEC was tested by MTT method to denote its cytotoxicity. The resultsproved that CS had no genotoxicity in the test dose range because there were no abnormalalterations of test bacteria or cells. CS could promote the proliferation and have nocytotoxicity in the test dose range (0.0625~1mg/mL).
(3) Reproductive, developmental and teratogenic toxicity: CS were injected into micebefore mating or on GD6, and the alterations in reproductive parameters, mating, fertilization,gestation, delivery, and lactation of F0mice were observed. The embryogenesis anddevelopment, growth and teratogenic conditions of offspring were recorded. The resultsshowed that there were no influences of CS in reproductive parameters of F0male mice,however, the corpora lutea counts and implantation sites of F0female mice were reducedsignificantly even at125mg/kg, which could lead to the reducing of the numbers of livefetuses. Therefore, the NOAEL of CS in the reproductive toxicity was considered to be lessthan125mg/kg. CS could induce emaciation and threatened abortion in F0mice at500mg/kg,and the numbers of live fetuses were decreased notably. However, the developments of itsoffspring were not affected. Therefore, the NOAEL of CS in the developmental toxicity wasconsidered to be125mg/kg. In addition, there was no difference in the teratogenic rate amongall groups, so the NOAEL of teratogenic toxicity was considered to be more than2000mg/kg.
In summary, CS, whose DD was85.32%and Mw was1.22×106, screened by in vitro andbody surface hemostatic tests and displayed good hemostatic activity in vivo, which couldsignificantly shorten the bleeding time and reduce the amount of bleeding. So it has a gooddevelopment and research prospect. The biological safety evaluation tests of CS as an internalhemostatic material were performed for the first time, and the results showed that the LD_(50) ofCS in mice was over5000mg/kg, and it had not shown genotoxicity, cytotoxicity orteratogenic toxicity. However, CS could induce some clinical adverse effects, especially in thereproductive process of female mouse and pregnant mouse. These results have importanttheoretical values and practical meaning for the development and application ofchitosan-based implantable hemostatic dressing.
为了筛选出具有良好止血活性的壳聚糖并评价其在体内的止血效果，利用试管法对比研究一系列具有不同脱乙酰度（DD）、分子量（Mw）的壳聚糖固体粉末和溶液在体外对兔血凝固时间的影响，利用小鼠断尾法测定不同性质壳聚糖粉末对鼠尾出血时间和出血量的影响，并将据此筛选得的壳聚糖样品应用于兔体内脏器的损伤创口，测定各创口的出血时间和出血量的变化。结果显示：壳聚糖样品的Mw和DD对其体外止血活性影响显著，Mw和DD越高，兔血凝固时间越短，且同等剂量下壳聚糖粉末的促凝血活性优于其溶液；将各壳聚糖粉末样品应用于小鼠断尾创口，出血时间均可缩短53.4%以上，DD为85.32%、Mw为1.22×106的壳聚糖粉末（CS）更可将出血时间缩短74.7%，并将出血量减少86.4%；将该CS粉末应用于动物体内脏器创口，最高可缩短出血时间63.5%、减少出血量72.9%，对体内的多血管实质性脏器（如肝、脾）的出血创口具有显著的止血效果。
为了探讨CS发挥体内止血活性的可能途径，将其与广泛分布于血管内腔表面的内皮细胞共培养24h，对于细胞合成与分泌的促凝血因子血管性假性血友病因子（vWF）、组织因子（TF）、纤溶酶原活化物抑制剂（PAI）、内皮素（ET-1）和抗凝血因子内皮型一氧化氮合成酶（ENOs）、血栓调节蛋白（TM）、蛋白S（PS）、组织型纤溶酶原活化物（tPA）、组织因子途径抑制因子（TFPI）等mRNA表达量的变化，用RT-PCR和荧光定量分析法进行测定。结果表明：CS对抗凝血因子TFPI、tPA和ENOs的mRNA表达均有一定的抑制作用，高剂量CS（1mg/mL）更可使其表达量分别降低至空白对照组的34.40%、0.77%和9.24%。因此，通过抑制内皮细胞抗凝血因子的合成分泌而使血液加速凝固，可能是CS体内止血的途径之一。
对于体内止血效果显著的CS的生物安全性，重点从以下几方面进行了研究。
(1)急性毒性和长期毒性：分别按照2000mg/kg和5000mg/kg的限度剂量于ICR小鼠腹腔注射CS混悬液，观察短期内动物的生存状况和不良反应症状，检查其血液学和组织学变化，以此评价其急性毒性；对小鼠腹腔注射CS混悬液后持续观察90d，根据动物的生存状况、不良反应、血液学和组织学检查结果评价其长期毒性。结果显示：腹腔注射CS混悬液的LD_(50)>5000mg/kg，属于低毒物质；小鼠出现体重和摄食量显著下降、毛发竖立、腹泻、活动量急剧减少等短期临床毒性反应，机体产生对外来物质的急性炎症反应，肝和肾有显著的炎症反应，脾脏因其白髓弥散性增生而显著增大；CS在小鼠体内的长期存在，可引起机体慢性炎症、脾脏持续增大和血小板数量持续增加。
(2)遗传毒性和细胞毒性：通过Ames实验、小鼠微核实验和精子畸形实验对CS的遗传毒性和致突变作用进行了研究，同时利用MTT法研究了CS对内皮细胞HUVEC增殖率的影响，以此表征其细胞毒性。结果显示：在实验剂量范围内，CS未引起测试菌株或细胞的异常变化，无显著的遗传毒性；CS可促进HUVEC增殖，在0.0625~1mg/mL范围内无细胞毒性。
(3)生殖毒性、发育毒性和致畸毒性：小鼠分别于交配前和妊娠早期（GD6）腹腔注射CS，观察亲代F0小鼠的生殖参数和交配、受精、妊娠、分娩及哺乳等过程的变化，记录其胚胎形成、发育及子代的生长和畸形状况。结果显示：CS对F0雄鼠的生殖参数未有显著影响，但低剂量CS（125mg/kg）即可引起F0雌鼠黄体数和着床数的显著下降，从而导致活胎数的减少，因此，CS对小鼠生殖毒性的无明显损害作用水平（NOAEL）低于125mg/kg；中剂量CS（500mg/kg）即可引起F0孕鼠消瘦和先兆流产，其活胎数显著减少，但其仔鼠的生长发育未受显著影响，因此，CS对小鼠发育毒性的NOAEL为125mg/kg；同时，各剂量组小鼠畸形率无显著差异，CS的致畸毒性NOAEL大于2000mg/kg。
综上所述，根据体外和动物体表止血实验筛选得DD为85.32%、Mw为1.22×106的壳聚糖样品CS，其应用于动物体内脏器创口时，可显著缩短出血时间并降低出血量，具有良好的体内止血效果，具有较好的开发和研究前景；首次对作为体内止血材料的壳聚糖样品进行系统的生物安全性评价，结果显示其在小鼠体内的LD_(50)>5000mg/kg，无遗传毒性、细胞毒性和致畸毒性，但是亦可引起一定的临床不良反应，尤其是对雌鼠的生殖和孕鼠的妊娠过程影响较大，该结果对于壳聚糖体内止血敷料的开发和应用具有重要的理论价值和指导意义。
Chitosan is a natural biodegradability polysaccharide with hemostasis and wound healingactivities. It has been a research highlight as a kind of hemostatic biomaterials in recent years,and there are several kinds of topical chitosan-based dressing approved for listing. However,the development and application of chitosan as an implanted hemostatic material are still intheir infancy, which is limited by its unclear hemostatic effect and biological safety in vivo.The purposes of this thesis are to evaluate the hemostatic capacity and biological safety ofchitosan in animals, to lay research foundations for its development and offer academic basesfor its application as an in vivo hemostatic dressing, and this has important theoretical andpractical values in the developments of chitosan industry.
In order to screen a sample with good hemostatic activity and evaluate its hemostaticcapability in vivo, the effects of a series of chitosan powder and solution with different DDand Mw to the coagulation time of rabbit blood in vitro were studied by Lee-White method,and the effects of chitosan powder samples with different properties to the time and theamount of bleeding of mouse tail were investigated by tail cutting method. The samplescreened according to these was applied to the organ wounds of rabbits in vivo, and the timeand amount of bleeding were measured. The results showed that the Mw and DD had greateffects on the hemostatic activity of chitosan, the coagulation time was shorter while the Mwand DD were higher, and the activity of powder was better than the solution at the same dose.All chitosan powder samples could shorten the bleeding time of mouse tail more than53.4%and CS powder, whose DD was85.32%and Mw was1.22×106, could reduce the bleedingtime by74.7%and the amount of bleeding by86.4%. CS powder showed a good hemostaticeffect on the bleeding wounds of substantive organs with rich blood (such as liver and spleen),while the bleeding time could be shortened63.5%and the amount reduced72.9%at most.
To explore the possible way of CS to play its hemostatic activity in vivo, it wasco-cultured with endothelial cells, which were widely distributed in the vessel lumen surface,for24h. The changes of procoagulant factors (such as vWF, TF, PAI, and ET-1) andanti-coagulant factors (such as ENOs, TM, PS, tPA, and TFPI) synthesized and secreted byendothelial cells were measured by RT-PCR and fluorescence quantitative analysis, whichcould denote the changes of coagulation capability of endothelial cells. The results showedthat CS could inhibited the mRNA expressions of TFPI, tPA, and ENOs, and their expressionamounts in the highest CS group were34.40%,0.77%, and9.24%of control group,respectively. Therefore, CS could promote blood clotting by inhibiting the synthesis andsecretion of anti-coagulant factors, and this might be one way of chitosan to stop the bleedingin vivo.
The key researches about the biological safety of CS were as follow.
(1) Acute and chronic toxicity: CS suspension was intraperitoneally injected into ICRmice at2000mg/kg and5000mg/kg, respectively. The survival condition and symptoms ofadverse effects were observed for14days, and hematological and histological tests wereperformed. Then CS suspension was singly injected intraperitoneally into ICR mice and the observation lasted for90days. The survival condition, symptoms of adverse effects,hematological and histological changes were observed to evaluate the chronic toxicity. Theresults showed that the LD_(50)of CS was over5000mg/kg in mice, and it was a less-toxicmaterial. Some adverse effects, such as decreasing of body weight and food consumption,erected hair, serious diarrhea, and sharply reduced movements, were observed. Acuteinflammations of body to strange materials were noted, inflammatory cells were increased inlivers and kidneys, and spleens were enlarged notably for the disseminated hyperplasis ofwhite pulp area. The long-term existence of CS in vivo could induce chronic inflammation ofbody, persistive enlargement of spleen, and continued increasement of platelet.
(2) Genotoxicity and cytotoxicity: the genotoxicity and mutagenicity of CS were studiedby Ames test, mouse micronucleus test and sperm abnormality test, and the effect of CS to theproliferation of HUVEC was tested by MTT method to denote its cytotoxicity. The resultsproved that CS had no genotoxicity in the test dose range because there were no abnormalalterations of test bacteria or cells. CS could promote the proliferation and have nocytotoxicity in the test dose range (0.0625~1mg/mL).
(3) Reproductive, developmental and teratogenic toxicity: CS were injected into micebefore mating or on GD6, and the alterations in reproductive parameters, mating, fertilization,gestation, delivery, and lactation of F0mice were observed. The embryogenesis anddevelopment, growth and teratogenic conditions of offspring were recorded. The resultsshowed that there were no influences of CS in reproductive parameters of F0male mice,however, the corpora lutea counts and implantation sites of F0female mice were reducedsignificantly even at125mg/kg, which could lead to the reducing of the numbers of livefetuses. Therefore, the NOAEL of CS in the reproductive toxicity was considered to be lessthan125mg/kg. CS could induce emaciation and threatened abortion in F0mice at500mg/kg,and the numbers of live fetuses were decreased notably. However, the developments of itsoffspring were not affected. Therefore, the NOAEL of CS in the developmental toxicity wasconsidered to be125mg/kg. In addition, there was no difference in the teratogenic rate amongall groups, so the NOAEL of teratogenic toxicity was considered to be more than2000mg/kg.
In summary, CS, whose DD was85.32%and Mw was1.22×106, screened by in vitro andbody surface hemostatic tests and displayed good hemostatic activity in vivo, which couldsignificantly shorten the bleeding time and reduce the amount of bleeding. So it has a gooddevelopment and research prospect. The biological safety evaluation tests of CS as an internalhemostatic material were performed for the first time, and the results showed that the LD_(50) ofCS in mice was over5000mg/kg, and it had not shown genotoxicity, cytotoxicity orteratogenic toxicity. However, CS could induce some clinical adverse effects, especially in thereproductive process of female mouse and pregnant mouse. These results have importanttheoretical values and practical meaning for the development and application ofchitosan-based implantable hemostatic dressing.
引文
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