本文選題：吸附透析 + 膽紅素　； 參考：《大連理工大學(xué)》2012年博士論文

【摘要】：分子吸附再循環(huán)系統(tǒng)(Molecular Adsorbents Recycling System, MARS)是目前臨床治療重癥肝病效果優(yōu)良的非生物型人工肝支持系統(tǒng)。在以人血清白蛋白(Human serum albumin, HSA)為分子吸附劑的吸附透析過程中,添加到透析液中的HSA與血液中的蛋白質(zhì)競爭性結(jié)合疏水性毒素并被透析液帶走,從而有效、廣譜去除患者血液中的蛋白質(zhì)結(jié)合毒素及水溶性毒素,實現(xiàn)改善患者體內(nèi)環(huán)境、促進肝臟功能恢復(fù)和為肝移植爭取時間的目的。MARS的治療效果已經(jīng)得到醫(yī)生和患者的廣泛認(rèn)可,在國外被推薦作為各類肝衰竭的標(biāo)準(zhǔn)治療方案。由于HSA是MARS實現(xiàn)解毒功能的關(guān)鍵分子吸附劑,用量較大,而作為注射制劑的HSA來源有限,價格昂貴。HSA來源受限及其再生設(shè)備復(fù)雜等問題導(dǎo)致MARS臨床治療費用高昂,其應(yīng)用人群范圍受限。鑒于此,本論文設(shè)計合成了一種水溶性膽紅素分子吸附劑替代HSA用于對肝病病人血液的吸附透析,該吸附劑在實現(xiàn)對膽紅素等毒素高效去除的同時,可以簡化治療設(shè)備,顯著降低治療成本。 在本論文中,首先針對膽紅素分子的結(jié)構(gòu)特點,選擇殼聚糖、右旋糖酐、聚乙烯亞胺(Polyethyleneimine, PEI)等水溶性高分子為載體,制備了帶有不同吸附功能基的系列膽紅素分子吸附劑。對其膽紅素吸附能力評價的結(jié)果表明：以PEI為載體、以β-環(huán)糊精(β-cyclodextrin,β-CD)為功能基的水溶性吸附劑P-CD-PEI在相同條件下對膽紅素的吸附透析能力最強。進一步以P-CD-PEI為研究對象,對其合成過程進行了優(yōu)化。最終獲得的β-CD-PEI的平均分子量約為157kD,平均每個β-CD-PEI分子上具有366個分枝點,偶聯(lián)β-CD的數(shù)量約為51個。選擇Nipro Sureflux-130G透析器,采用200mL含1%(w/v) p-CD-PEI的透析液對150mL含300mg／L膽紅素的人血漿進行吸附透析,其對血漿中膽紅素的1h吸附量達到3.55mg／g。 采用分子模擬的方法研究了分子吸附劑功能基p—CD與膽紅素的結(jié)合方式以及結(jié)合能,并與α-CD、γ-CD及HSA與膽紅素的相互作用進行了比較。研究結(jié)果表明：在環(huán)糊精家族中,α-CD由于疏水內(nèi)腔太小,不能與膽紅素形成包合物；β-CD及Y-CD與膽紅素形成的2：1包合物比1：1包合物更穩(wěn)定,其中p—CD膽紅素2：1包合物穩(wěn)定性高于HSA與膽紅素的復(fù)合物,意味著β-CD與膽紅素的結(jié)合能力強于HSA,有能力與HSA競爭結(jié)合膽紅素。偶聯(lián)反應(yīng)對β-CD分子結(jié)構(gòu)的改變不會減弱其與膽紅素的結(jié)合能力。進一步通過將α、β、γ-CD分別偶聯(lián)到PEI載體上合成了三種分子吸附劑,吸附透析實驗結(jié)果表明β-CD-PEI對膽紅素的結(jié)合能力最強；使用Benesi-Hildebrand法分析β-CD與膽紅素包合后膽紅素紫外可見光譜變化,進一步確認(rèn)了β-CD與膽紅素以2：1的比例形成包合物,從而證實了分子模擬研究的結(jié)果。 在吸附透析治療中,對血漿膽紅素的去除效果取決于透析膜的特性、血漿流速、透析液流速、血漿中的膽紅素濃度、透析液中水溶性吸附劑的濃度以及吸附透析時間等因素。在吸附透析系統(tǒng)的參數(shù)優(yōu)化中發(fā)現(xiàn)：當(dāng)血漿及透析液流速分別為300和50mL/min時,β-CD-PEI吸附透析對血漿膽紅素的去除效果最佳；超濾形成的跨膜液流產(chǎn)生的濃差極化現(xiàn)象增加了膽紅素的傳質(zhì)阻力,降低了β-CD-PEI在透析膜內(nèi)的分子擴散,不利于β-CD-PEI吸附透析對膽紅素的去除；以聚醚砜為膜材質(zhì)的Lengthen LST140透析器對于膽紅素的傳質(zhì)效果強于以三醋酸纖維素為膜材質(zhì)的Nipro Sureflux-130G透析器；透析膜表面及孔道中吸附的白蛋白能夠?qū)δ懠t素進行協(xié)助傳遞,進而提高透析膜對膽紅素的傳質(zhì)效果。 在吸附透析過程中,β-CD-PEI的吸附量隨著血漿中膽紅素濃度的升高而增加,采用1L濃度為1%的β-CD-PEI透析液對200mL膽紅素初始濃度分別為80.3、140.4、214.8、267.3、305.2mg／L的肝病病人血漿進行吸附透析,6h的總膽紅素去除率均能達到30-40%,適用于不同患病程度的肝病病人。增加分子吸附劑β-CD-PEI的用量可以提高吸附透析對膽紅素的去除效果。使用1L4%的β-CD-PEI透析液可以去除血漿中44.8%的膽紅素(初始濃度140.4mg／L),比相同情況下4L1%分子吸附劑的膽紅素去除率提高9個百分點。β-CD-PEI對血漿膽紅素的清除率比相同質(zhì)量分?jǐn)?shù)的牛血清白蛋白高9.5個百分點,同時β-CD-PEI對血漿中的總膽汁酸、芳香族氨基酸等疏水毒素也具有明顯的去除能力,說明β-CD-PEI具有替代白蛋白進行吸附透析的潛力。 通過基于物料衡算的數(shù)學(xué)模型描述了吸附透析過程中血漿膽紅素濃度變化規(guī)律。通過實驗數(shù)據(jù)回歸,計算得到了Nipro Sureflux-130G血液透析膜對膽紅素的總傳質(zhì)系數(shù)Dt為1.7L/min, p-CD-PEI與膽紅素的吸附平衡常數(shù)Ka1為22.7L/μmoL。進一步使用該模型預(yù)測了同一體系中血漿膽紅素初始濃度和吸附劑用量改變時膽紅素的吸附透析效果,平均誤差5%。 上述研究結(jié)果證明,膽紅素水溶性分子吸附劑β-CD-PEI對肝病患者血漿中的膽紅素及其它疏水性毒素具有較高的清除能力,同時具有價格低廉、治療設(shè)備簡單的優(yōu)勢。具有替代白蛋白應(yīng)用于臨床治療的潛力。
[Abstract]:Molecular Adsorbents Recycling System (MARS) is an excellent abiotic artificial liver support system for the treatment of severe liver disease. In the process of adsorption and dialysis with human serum albumin (Human serum albumin, HSA) as a molecular adsorbent, HSA and protein in the blood are added to the dialysate. Competitive combination of hydrophobic toxin and dialysate is taken away, which is effective and broad-spectrum removal of protein binding toxins and water-soluble toxins in the patient's blood to improve the environment of the patient, promote the recovery of liver function and the time for liver transplantation for the purpose of.MARS treatment has been widely recognized by doctors and patients abroad. It is recommended as the standard treatment for all kinds of liver failure. Because HSA is a key molecular adsorbent for MARS to realize detoxification, the amount of HSA is limited, the limited source of.HSA and the complexity of the regenerating equipment, such as the limited source of the price and the complexity of the regeneration equipment, are very expensive for the clinical treatment of MARS, and the scope of its application is limited. In view of this, In this paper, a water soluble bilirubin molecular adsorbent is designed and synthesized to replace HSA for the absorption and dialysis of the blood of patients with liver disease. The adsorbent can be used to effectively remove the bilirubin and other toxins. It can simplify the treatment equipment and reduce the cost of treatment significantly.
In this paper, a series of bilirubin molecular adsorbents with different adsorption functional groups were prepared by selecting the water soluble polymers such as chitosan, dextran, Polyethyleneimine, PEI, and other water-soluble polymers, such as chitosan, dextran, and polyethyleneimine (PEI). The results of the evaluation of the adsorption capacity of the bilirubin showed that PEI was used as the carrier. The adsorption and dialysis ability of beta cyclodextrin (beta -cyclodextrin, beta -CD) as a functional group, P-CD-PEI, was the strongest for bilirubin under the same condition. The synthesis process was optimized with P-CD-PEI as the research object. The average molecular weight of beta -CD-PEI was about 157kD, with an average of 366 on each beta -CD-PEI molecule. At the branch point, the number of the coupling beta -CD was about 51. The Nipro Sureflux-130G dialyzer was selected and the 200mL containing 1% (w/v) p-CD-PEI dialysate was used to adsorb the human plasma containing 300mg / L bilirubin in 150mL, and the 1H adsorption capacity of the bilirubin in plasma reached 3.55mg / g..
The binding energy and binding energy of the functional group P CD with bilirubin are studied by molecular simulation, and the interaction between the alpha -CD, gamma -CD and HSA with bilirubin is compared. The results show that in the cyclodextrin family, the alpha -CD can not form a inclusion complex with bilirubin because the hydrophobic inner cavity is too small; beta -CD and Y-CD are found in the cyclodextrin family. The 2:1 inclusion complex formed by bilirubin is more stable than the 1:1 inclusion complex, and the stability of the p - CD bilirubin 2:1 inclusion complex is higher than that of the HSA and bilirubin complex, which means that the binding ability of beta -CD to bilirubin is stronger than that of HSA, and it has the ability to compete with the bilirubin in competition with HSA. The change of the coupling reaction to the molecular structure of beta -CD does not weaken the binding of bilirubin to the bilirubin. Three kinds of molecular adsorbents were synthesized by coupling the alpha, beta, and gamma -CD to the PEI carrier. The adsorption dialysis experiment results showed that the binding ability of beta -CD-PEI to bilirubin was the strongest. Benesi-Hildebrand method was used to analyze the visible spectrum changes of bilirubin after beta -CD and bilirubin inclusion, and further confirmed the beta -CD and bilirubin. The inclusion complex was formed in the ratio of 2:1, which confirmed the result of molecular simulation.
In the treatment of adsorption dialysis, the removal of plasma bilirubin depends on the characteristics of the dialysis membrane, the flow velocity of the plasma, the flow velocity of dialysate, the concentration of bilirubin in the plasma, the concentration of water soluble adsorbents in the dialysate and the time of adsorption dialysis. For 300 and 50mL/min, the removal of plasma bilirubin by beta -CD-PEI adsorption was the best. The concentration polarization caused by the ultrafiltration of the transmembrane fluid increased the mass transfer resistance of bilirubin, reduced the molecular diffusion of beta -CD-PEI in the dialysis membrane, and was not conducive to the removal of bilirubin by beta -CD-PEI adsorption dialysis; polyethersulfone was used as the membrane material. The mass transfer effect of Lengthen LST140 dialyzer on bilirubin is better than that of the Nipro Sureflux-130G dialyzer with three cellulose acetate membrane. The albumin adsorbed on the surface of the dialysis membrane and the pore channel can assist the transmission of bilirubin, and then improve the quality of the dialysate to bilirubin.
In the process of adsorption dialysis, the adsorption amount of beta -CD-PEI increases with the increase of the concentration of bilirubin in plasma. The plasma of the patients with the initial concentration of 200mL bilirubin is 80.3140.4214.8267.3305.2mg / L with the concentration of 1L 1%, and the removal rate of total bilirubin in 6h can reach 30-40%, and the application of the total bilirubin removal rate of 6h can reach 30-40%. Increasing the dosage of molecular adsorbent beta -CD-PEI can improve the removal of bilirubin by adsorption dialysis. The use of 1L4%'s beta -CD-PEI dialysate can remove 44.8% of the bilirubin (initial concentration 140.4mg / L) in plasma, and increase the bilirubin removal rate by 9 percent than that of 4L1% sub sorbents under the same condition. The scavenging rate of beta -CD-PEI to plasma bilirubin is 9.5 percentage points higher than that of the same mass fraction of bovine serum albumin, while beta -CD-PEI also has the ability to remove hydrophobic toxins such as total bile acid and aromatic amino acids in plasma, indicating that beta -CD-PEI has the potential to replace albumin for adsorption dialysis.
The change of plasma bilirubin concentration in the process of adsorption dialysis was described by a mathematical model based on material balance. The total mass transfer coefficient of Nipro Sureflux-130G hemodialysis membrane to bilirubin was calculated to be 1.7L/min, and the adsorption equilibrium constant Ka1 of p-CD-PEI and bilirubin was further used for 22.7L/ u moL. by regression of experimental data. The model predicted the adsorption dialysis effect of bilirubin in the same system when the initial concentration of plasma bilirubin and the dosage of adsorbent changed. The average error was 5%.
The above results show that bilirubin water soluble molecular adsorbent beta -CD-PEI has high scavenging ability for bilirubin and other hydrophobic toxins in plasma of patients with liver disease, and has the advantages of low price and simple treatment equipment. It has the potential to replace albumin in clinical treatment.
【學(xué)位授予單位】：大連理工大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R318.0

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