CN105535946A - Application of transglutaminase in strengthening corneal mechanical properties and biological preparation - Google Patents
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/45—Transferases (2)
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- C12Y—ENZYMES
- C12Y203/00—Acyltransferases (2.3)
- C12Y203/02—Aminoacyltransferases (2.3.2)
- C12Y203/02013—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
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Abstract
本发明针对现有的角膜交联技术的不足,提供一种新的可以明显加强角膜力学性质的生物制剂,该制剂为具有交联作用的生物酶(转谷氨酰胺酶),可用于加强角膜生物力学性质。该生物制剂可以通过催化角膜基质中胶原纤维中特定氨基酸,产生异肽键,加强胶原纤维的连接,从而使角膜基质中各层纤维小板之间的相互连接增强,到达增强角膜基质的力学性质的目的。采用本发明的生物制剂介导交联后眼角膜全层的基质细胞活性仍较好,未出现明显的细胞凋亡,安全性较高。相比目前临床使用的角膜交联的治疗方案,本发明具有更好的交联效果和更少的副作用和并发症,具有很强的临床应用前景。The present invention aims at the deficiencies of the existing corneal cross-linking technology, and provides a new biological preparation that can obviously strengthen the mechanical properties of the cornea. The preparation is a biological enzyme (transglutaminase) with cross-linking effect, which can be used to strengthen the cornea biomechanical properties. The biological agent can catalyze specific amino acids in the collagen fibers in the corneal stroma to generate isopeptide bonds and strengthen the connection of collagen fibers, thereby enhancing the interconnection between the fiber platelets in the corneal stroma and achieving the enhancement of the mechanical properties of the corneal stroma the goal of. After the cross-linking is mediated by the biological agent of the present invention, the activity of the stromal cells in the whole layer of the cornea is still good, no obvious cell apoptosis occurs, and the safety is high. Compared with the currently clinically used corneal cross-linking treatment plan, the present invention has better cross-linking effect and fewer side effects and complications, and has a strong clinical application prospect.
Description
技术领域 technical field
本发明涉及转谷氨酰胺酶在加强角膜力学性质中的应用及其制备的生物制剂,属于生物制剂/角膜病领域。 The invention relates to the application of transglutaminase in enhancing the mechanical properties of cornea and the biological preparation prepared therefrom, belonging to the field of biological preparations/corneal diseases.
背景技术 Background technique
圆锥角膜(Keratoconus)是一种不伴有炎症、以角膜基质变薄、角膜进行性前突、角膜不规则散光、矫正视力下降为特征的眼病。也是角膜扩张类疾病的典型代表。 Keratoconus is a non-inflammatory eye disease characterized by thinning of the corneal stroma, progressive corneal protrusion, irregular corneal astigmatism, and decreased corrected visual acuity. It is also a typical representative of corneal ectasia.
圆锥角膜多发生于年轻人,发病率约为1/2000,约21%的圆锥角膜患者最终需采用角膜移植手术治疗,约占所有角膜移植患者16%。传统的圆锥角膜的主要治疗有屈光矫正和角膜移植两大类,前者包括配戴框架眼镜和角膜接触镜、角膜内环植入术、准分子激光角膜表面切削术(PRK)等,这些方法对部分圆锥角膜患者可达到屈光矫正的目的,但不能阻止病情的进展。后者则受到器官来源的限制,患者需要使用抗排异药物,应用受到制约。 Keratoconus mostly occurs in young people, and the incidence rate is about 1/2000. About 21% of keratoconus patients eventually need corneal transplantation, accounting for about 16% of all corneal transplantation patients. The traditional main treatments for keratoconus include refractive correction and corneal transplantation. The former includes wearing frame glasses and contact lenses, intracorneal ring implantation, excimer laser keratectomy (PRK), etc. These methods For some patients with keratoconus, the purpose of refractive correction can be achieved, but the progression of the disease cannot be prevented. The latter is limited by the source of organs, and patients need to use anti-rejection drugs, so the application is restricted.
圆锥角膜的病因至今仍不十分清楚,但角膜的机械强度降低被认为是其重要病因之一。组织学上发现圆锥处纤维板层减少,胶原纤维直径并未改变。故认为可能是纤维板层间粘合不够,板层相互滑脱,导致角膜变薄。继而,由于角膜中央区或旁中央区进行性的变薄,导致角膜不规则散光度增加,近视度数也不断加深,视力进行性下降。从这个角度出发,增加角膜的力学强度成为治疗该疾病的新的治疗手段和思路。 The cause of keratoconus is still not very clear, but the reduction of the mechanical strength of the cornea is considered to be one of its important causes. Histologically, the fibrolamellar layer of the cone was found to be reduced, and the diameter of the collagen fibers was not changed. Therefore, it is considered that the adhesion between the fiberboard layers may be insufficient, and the layers of the fiberboards slip off each other, resulting in thinning of the cornea. Then, due to the progressive thinning of the central or paracentral area of the cornea, the degree of irregular astigmatism of the cornea increases, the degree of myopia continues to deepen, and the visual acuity decreases progressively. From this point of view, increasing the mechanical strength of the cornea has become a new treatment method and idea for the treatment of this disease.
胶原交联治疗(cross-linkingtreatment)是通过生物化学的方法使胶原纤维的相互连接加强,增加组织强度的治疗技术,按照交联方法的不同可以分为物理交联(光化学交联)、化学交联、生化交联(酶交联)三种。在目前国内外的研究中,物理交联(光化学交联)治疗对圆锥角膜的治疗研究已经显示了很好的疗效。其中紫外光-核黄素角膜交联法已经被美国FDA批准,在临床上应用。 Collagen cross-linking treatment (cross-linking treatment) is a treatment technique that strengthens the interconnection of collagen fibers and increases tissue strength through biochemical methods. According to different cross-linking methods, it can be divided into physical cross-linking (photochemical cross-linking) and chemical cross-linking. There are three types of cross-linking and biochemical cross-linking (enzyme cross-linking). In the current research at home and abroad, physical cross-linking (photochemical cross-linking) therapy has shown a good effect on the treatment of keratoconus. Among them, the ultraviolet light-riboflavin corneal cross-linking method has been approved by the US FDA and is used clinically.
该方法是采用核黄素作为光敏剂,以对眼球内部结构无害的紫外线A(UVA)照射,利用光线的照射使光敏剂将氧分子激发到三线态,其在回到单线态时产生有很强的氧化活性的氧自由基(ROS,reactiveoxygenspecies)(图6),使得胶原纤维的终端发生缩合反应,增加了胶原纤维之间的侧向连接,从而使角膜的机械强度得到了增强,圆锥角膜得以停止进展甚至有部分患者近视度数得到了减轻。化学交联对角膜的作用目前停留在实验室阶段,有学者使用短链无环β硝基乙醇(BNA)、京尼平用于猪角膜的交联,发现离体动物角膜的弹性模量一定程度的增加。 The method is to use riboflavin as a photosensitizer to irradiate with ultraviolet A (UVA) which is harmless to the internal structure of the eyeball, and utilize the irradiation of light to make the photosensitizer excite the oxygen molecules to the triplet state, which will generate active oxygen molecules when returning to the singlet state. Strong oxidative activity of oxygen free radicals (ROS, reactiveoxygenspecies) (Figure 6) makes the terminals of collagen fibers undergo condensation reactions, increasing the lateral connection between collagen fibers, thereby enhancing the mechanical strength of the cornea, and the cone The progress of the cornea can be stopped and even the myopia degree of some patients has been alleviated. The effect of chemical cross-linking on the cornea is currently in the laboratory stage. Some scholars used short-chain acyclic β-nitroethanol (BNA) and genipin to cross-link pig corneas, and found that the elastic modulus of isolated animal corneas was constant. degree of increase.
上述两种交联方法均存在一定的局限性。 Both of the above-mentioned cross-linking methods have certain limitations.
虽然紫外光-核黄素交联法技术应用于临床已近十年,并被誉为“圆锥角膜患者的治愈希望”。但是尚存在诸多不足:1)由于角膜的核黄素吸收效应,该方法只能使角膜前250~300um的基质胶原得到交联,因此,其临床有效性受限,多项研究均显示紫外光核黄素交联后角膜最陡屈光度下降仅2D左右。2)该方法治疗时需去除角膜上皮,可增加患者术后疼痛、视物模糊、角膜云雾状混浊及感染的发生。3)该方法也存在安全性问题,近期临床研究报道对角膜厚度低于400um的圆锥角膜患者行紫外线核黄素交联后角膜内皮细胞密度明显下降。4)对基质细胞的损伤:基础研究及角膜共聚焦显微镜临床观察均表明在深达300um的角膜基质中有大量角膜细胞凋亡,需要长达半年时间恢复。5)紫外光核黄素胶原交联对于光过敏体质的病人不适用。6)设备价格昂贵。7)对于术前最陡角膜屈光度超过58D、年龄超过35岁、视力大于0.8的圆锥角膜患者,紫外光核黄素交联手术后发生各种并发症的风险明显增加。上述问题限制了此项技术的推广和应用。化学交联法简单易行,交联剂来源广,但是其化学交联剂治疗的副作用难以回避、其对眼部其他组织的生物毒性需要阐明。相比之下,生化交联(酶交联)成为一个比较理想的选择。 Although the ultraviolet light-riboflavin cross-linking method has been used clinically for nearly ten years, it is known as "the hope of curing keratoconus patients". However, there are still many deficiencies: 1) Due to the riboflavin absorption effect of the cornea, this method can only cross-link the stromal collagen of the anterior 250-300um of the cornea. Therefore, its clinical effectiveness is limited, and many studies have shown that ultraviolet light After riboflavin cross-linking, the steepest corneal diopter drop was only about 2D. 2) This method needs to remove the corneal epithelium during treatment, which can increase the occurrence of postoperative pain, blurred vision, corneal cloudy turbidity and infection. 3) This method also has safety issues. Recent clinical studies have reported that corneal endothelial cell density decreased significantly after ultraviolet riboflavin cross-linking in keratoconus patients with corneal thickness less than 400um. 4) Damage to stromal cells: Basic research and clinical observation of corneal confocal microscopy have shown that a large number of keratocytes are apoptotic in the corneal stroma as deep as 300um, and it takes up to half a year to recover. 5) Ultraviolet light riboflavin collagen cross-linking is not suitable for patients with photosensitivity. 6) The equipment is expensive. 7) For patients with keratoconus whose preoperative steepest corneal diopter exceeds 58D, age over 35 years, and visual acuity greater than 0.8, the risk of various complications after ultraviolet riboflavin cross-linking surgery is significantly increased. The above problems limit the popularization and application of this technology. The chemical cross-linking method is simple and easy, and the sources of cross-linking agents are wide, but the side effects of the chemical cross-linking agent treatment are unavoidable, and its biological toxicity to other ocular tissues needs to be clarified. In contrast, biochemical cross-linking (enzyme cross-linking) becomes an ideal choice.
生化交联是利用有活性的酶,催化特定的氨基酸底物,使得肽链的氨基酸之间产生共价键,达到交联的目的。转谷氨酰胺酶(TGase)就是一种优秀的交联酶。 Biochemical cross-linking is the use of active enzymes to catalyze specific amino acid substrates to generate covalent bonds between amino acids in the peptide chain to achieve the purpose of cross-linking. Transglutaminase (TGase) is an excellent cross-linking enzyme.
目前发现的TGase共包括8个亚型,其中多数类型在人体中广泛存在,并参与重要的生理功能。TGase的生物学功能是催化蛋白质谷氨酸的酸残基和赖氨酸的铵残基之间发生转谷氨酰胺反应,生成异肽键:ε-(γ-谷氨酰)-赖氨酸键(图2)。该异肽键形成后异常稳定,可有效地增加组织对物理、化学的抵抗力,从而增加组织的力学强度。 The TGases discovered so far include 8 subtypes, most of which widely exist in the human body and participate in important physiological functions. The biological function of TGase is to catalyze the transglutamination reaction between the acid residue of protein glutamic acid and the ammonium residue of lysine to generate an isopeptide bond: ε-(γ-glutamyl)-lysine key (Figure 2). The isopeptide bond is extremely stable after formation, which can effectively increase the physical and chemical resistance of the tissue, thereby increasing the mechanical strength of the tissue.
该反应具有专一性、高效性的特点;酶催化反应作为一种常见的生理现象,在局部组织使用产生的组织损伤较小;使用酶催化反应,其所需的设备简单,操作简单,具有推广价值。 The reaction has the characteristics of specificity and high efficiency; as a common physiological phenomenon, enzyme-catalyzed reaction causes less tissue damage when used in local tissues; the use of enzyme-catalyzed reaction requires simple equipment, simple operation, and Promote value.
角膜基质的主要组成部分,占角膜全厚的90%以上,是角膜力学结构稳定的主要因素。其包括200-250层平行排列的纤维小板,主要成分为I型胶原纤维。I型胶原纤维是包括两条α1(I)肽链和一条α2(I)肽链的螺旋结构的大分子蛋白,其中α1(I)肽链的1464个氨基酸中,包括赖氨酸57个(5.24%),谷氨酸75个(6.94%),α2(I)肽链的1366个氨基酸中,包括赖氨酸50个(4.95%),谷氨酸66个(6.58%)。故总体上看,角膜基质具有5%以上的赖氨酸和谷氨酸,可以成为TGase催化反应的底物。 The main component of the corneal stroma, which accounts for more than 90% of the total thickness of the cornea, is the main factor for the stability of the corneal mechanical structure. It includes 200-250 layers of fibrous platelets arranged in parallel, and the main component is type I collagen fibers. Type I collagen fibers are macromolecular proteins with a helical structure including two α1(I) peptide chains and one α2(I) peptide chain, in which 1464 amino acids of the α1(I) peptide chain include 57 lysines ( 5.24%), 75 glutamic acids (6.94%), 1366 amino acids in the α2 (I) peptide chain, including 50 lysines (4.95%), 66 glutamic acids (6.58%). Therefore, in general, the corneal stroma has more than 5% lysine and glutamic acid, which can be the substrate of TGase catalytic reaction.
发明内容 Contents of the invention
本发明的目的是针对上述现有治疗圆锥角膜技术的不足,提供一种可以明显加强角膜力学性质的生物制剂,尤其是一种含有转谷氨酰胺酶的生物制剂在加强角膜力学性质方面的应用,转谷氨酰胺酶可以催化角膜基质中的赖氨酸和谷氨酸的交联反应,使角膜可以达到有效交联,力学性质得到明显加强。 The purpose of the present invention is to address the above-mentioned deficiencies in the existing technology for treating keratoconus, and to provide a biological preparation that can significantly enhance the mechanical properties of the cornea, especially the application of a biological preparation containing transglutaminase in enhancing the mechanical properties of the cornea , transglutaminase can catalyze the cross-linking reaction of lysine and glutamic acid in the corneal stroma, so that the cornea can achieve effective cross-linking, and the mechanical properties are significantly enhanced.
本发明一方面提供一种用于加强角膜力学性质的生物制剂,包括转谷氨酰胺酶和药用载体。 One aspect of the present invention provides a biological agent for enhancing the mechanical properties of the cornea, including transglutaminase and a pharmaceutical carrier.
其中,所述转谷氨酰胺酶用于催化角膜基质中的赖氨酸和谷氨酸的交联反应。 Wherein, the transglutaminase is used to catalyze the cross-linking reaction of lysine and glutamic acid in corneal stroma.
特别是,所述转谷氨酰胺酶可来源于购买或采用现有技术制备得到。 In particular, the transglutaminase can be purchased or prepared using existing technologies.
尤其是,通过将所述转谷氨酰胺酶和药用载体进行融合,形成眼药水形态的生物制剂。 In particular, the biological preparation in the form of eye drops is formed by fusing the transglutaminase with a pharmaceutical carrier.
特别是,通过将所述转谷氨酰胺酶和药用载体进行融合,形成眼药膏形态的生物制剂。 In particular, the biological preparation in the form of eye ointment is formed by fusing the transglutaminase with a pharmaceutical carrier.
本发明另一方面提供一种转谷氨酰胺酶在加强角膜力学性质中的应用。 Another aspect of the present invention provides an application of transglutaminase in enhancing corneal mechanical properties.
角膜基质的主要组成部分包括200-250层平行排列的纤维小板,是决定角膜力学结构稳定的主要因素,纤维小板的主要成分为I型胶原纤维,I型胶原纤维的肽链中包括5%以上的赖氨酸和谷氨酸。而转谷氨酰胺酶通过催化角膜基质中的赖氨酸和谷氨酸的交联反应,使各层纤维小板之间通过氨基酸反应相互交联,从而增强了角膜基质的力学性质。 The main components of the corneal stroma include 200-250 layers of fiber platelets arranged in parallel, which is the main factor determining the stability of the corneal mechanical structure. The main component of the fiber platelets is type I collagen fibers, and the peptide chain of type I collagen fibers includes 5 % above lysine and glutamic acid. Transglutaminase, by catalyzing the cross-linking reaction of lysine and glutamic acid in the corneal stroma, makes the fiber platelets of each layer cross-link each other through amino acid reactions, thereby enhancing the mechanical properties of the corneal stroma.
本发明的优点和有益技术效果如下: Advantage of the present invention and beneficial technical effect are as follows:
1、本发明转谷氨酰胺酶通过催化角膜基质中的赖氨酸和谷氨酸的交联反应,使角膜基质中各层纤维小板之间通过氨基酸反应相互交联,从而增强了角膜基质的力学性质。 1. The transglutaminase of the present invention catalyzes the cross-linking reaction of lysine and glutamic acid in the corneal stroma, so that the fiber platelets in each layer of the corneal stroma are cross-linked by amino acid reactions, thereby strengthening the corneal stroma mechanical properties.
2、采用本发明转谷氨酰胺酶介导交联的眼角膜的杨氏模量较未采用本发明转谷氨酰胺酶介导交联的眼角膜的杨氏模量平均提升了44%,应力应变曲线明显变陡,说明经转谷氨酰胺酶生化交联后,角膜的力学性质明显加强。 2. The Young's modulus of the cornea that uses the transglutaminase of the present invention to mediate the cross-linking is increased by 44% on average compared with the Young's modulus of the cornea that does not adopt the transglutaminase of the present invention to mediate the cross-linking, The stress-strain curve became significantly steeper, indicating that the mechanical properties of the cornea were significantly enhanced after transglutaminase biochemical cross-linking.
3、采用本发明转谷氨酰胺酶介导交联后眼角膜全层的基质细胞活性仍较好,未出现明显的细胞凋亡,说明本发明转谷氨酰胺酶对眼角膜基质基本无损伤。 3. The activity of stromal cells in the full thickness of the cornea after cross-linking mediated by the transglutaminase of the present invention is still good, and no obvious cell apoptosis occurs, indicating that the transglutaminase of the present invention has basically no damage to the corneal stroma .
附图说明 Description of drawings
图1为实施例1中研究眼和对照眼的应力应变曲线图; Fig. 1 is the stress-strain curve figure of research eye and contrast eye in embodiment 1;
图2位实施例2中对照组角膜基质的TUNEL染色结果图; The TUNEL staining result figure of control group corneal stroma in Fig. 2 embodiment 2;
图3为实施例2中生化交联组角膜基质的TUNEL染色结果图; Fig. 3 is the TUNEL staining result figure of biochemical cross-linking group corneal stroma in embodiment 2;
图4位实施例2中对照组角膜基质的DAPI染色结果图; The DAPI staining result figure of control group corneal stroma in Fig. 4 bit embodiment 2;
图5位实施例2中生化交联组角膜基质的DAPI染色结果图; The DAPI staining result diagram of the corneal stroma of the biochemical cross-linking group in Fig. 5;
图6为核黄素-紫外光交联原理示意图; Fig. 6 is a schematic diagram of riboflavin-ultraviolet light crosslinking principle;
图7为TGase催化缩合反应及异肽键生成图。 Figure 7 is a diagram of TGase catalyzed condensation reaction and formation of isopeptide bonds.
具体实施方式 detailed description
本发明实施例中转谷氨酰胺酶为德国Ruibio公司提供转谷氨酰胺酶,2.5KU/25g包装;新西兰大白兔为北大医院实验动物房提供购自芳元缘公司的试验用新西兰大白兔,体重2.5-3.0kg,该动物实验符合《中华人民共和国实验动物管理条例》和NIH《实验动物饲养管理和使用指南》的有关规定。 Transglutaminase in the embodiment of the present invention provides transglutaminase for German Ruibio company, 2.5KU/25g packing; 2.5-3.0kg, the animal experiment complies with the relevant provisions of the "Regulations of the People's Republic of China on the Administration of Experimental Animals" and the NIH "Guidelines for the Feeding, Management and Use of Experimental Animals".
实施例1转谷氨酰胺酶对兔角膜交联效果实验 Embodiment 1 Transglutaminase cross-linking effect experiment on rabbit cornea
取12只新西兰大白兔,以其右眼为研究眼,研究眼采用0.8U转谷氨酰胺酶介导的生化交联反应30min,饲养2周后,观察其角膜性质;以其左眼为对照眼。分别测定研究眼和对照眼的杨氏模量,结果见表1。分别测定研究眼和对照眼的应力应变,绘制应力应变曲线,见图1。 Take 12 New Zealand white rabbits, take the right eye as the research eye, use 0.8U transglutaminase-mediated biochemical cross-linking reaction for 30min in the research eye, and observe the corneal properties after feeding for 2 weeks; use the left eye as the control Eye. The Young's modulus of the study eye and the control eye were measured respectively, and the results are shown in Table 1. The stress-strain of the study eye and the control eye were measured respectively, and the stress-strain curve was drawn, as shown in Figure 1.
表1TGase对兔角膜交联效果 Table 1 TGase cross-linking effect on rabbit cornea
由表1可以看出,研究组角膜的杨氏模量较对照组平均提升了44%,由图1可以看出,研究组(OS)角膜的应力应变曲线较对照组(OD)变陡,说明相对于对照组,研究组角膜的应力由明显提升,因此可以得出,经转谷氨酰胺酶生化交联后,角膜的力学性质明显加强。 It can be seen from Table 1 that the Young's modulus of the study group cornea increased by 44% on average compared with the control group. It can be seen from Figure 1 that the stress-strain curve of the study group (OS) cornea was steeper than that of the control group (OD). It shows that compared with the control group, the stress of the cornea in the study group is significantly increased. Therefore, it can be concluded that the mechanical properties of the cornea are significantly enhanced after transglutaminase biochemical cross-linking.
实施例2转谷氨酰胺酶生化交联对角膜基质的损伤测试 Example 2 The damage test of transglutaminase biochemical cross-linking to corneal stroma
取12只新西兰大白兔,分为两组,每组6只,其中一组为生化交联组,采用0.8U转谷氨酰胺酶介导的生化交联反应30min,饲养2周后,观察其角膜基质细胞的凋亡;另一组为对照组,采用紫外光-核黄素交联,饲养2周后,观察其角膜基质细胞的凋亡。不同组白兔角膜基质的TUNEL染色结果见图2和图3,不同组白兔角膜基质的DAPI染色结果见图4和图5。 Take 12 New Zealand white rabbits and divide them into two groups, 6 rabbits in each group. One group is the biochemical cross-linking group. The biochemical cross-linking reaction mediated by 0.8U transglutaminase is used for 30 minutes. After feeding for 2 weeks, observe the Apoptosis of corneal stromal cells; the other group was the control group, which was cross-linked with ultraviolet light-riboflavin. After feeding for 2 weeks, the apoptosis of corneal stromal cells was observed. The results of TUNEL staining of corneal stroma of different groups of white rabbits are shown in Figure 2 and Figure 3, and the results of DAPI staining of corneal stroma of white rabbits of different groups are shown in Figure 4 and Figure 5.
图2为对照组的角膜,可以看出前1/2基质均出现细胞凋亡,图3为生化交联组的角膜,可以看出角膜未出现明显的细胞凋亡。图4为对照组的角膜,可以看出前半部角膜基质细胞活性已经大大降低,图5为生化交联组的角膜,可以看出角膜全层的基质细胞活性仍较好。 Figure 2 is the cornea of the control group, and it can be seen that the front 1/2 stroma has apoptosis, and Figure 3 is the cornea of the biochemical cross-linking group, and it can be seen that the cornea has no obvious apoptosis. Figure 4 shows the cornea of the control group. It can be seen that the activity of the corneal stromal cells in the first half has been greatly reduced. Figure 5 shows the cornea of the biochemical cross-linking group.
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