【作者】 李秋实；

【导师】 周延民；

【作者基本信息】 吉林大学 ， 口腔临床医学， 2010， 博士

【摘要】 激光是一种新型光源,具有方向性好、亮度高、单色性好、相干性好等优异的物理特性,可以在医学的诊断和治疗方面发挥巨大作用。低能量激光治疗(Low-Level-Laser- Therapy,LLLT)具有生物刺激效应,学者们发现它可以减轻疼痛、炎症、水肿,促进伤口、深层组织和神经的愈合。已有大量的体外和动物实验的研究证实LLLT可以促进成骨细胞增殖、分化和骨组织愈合,但应用于口腔种植领域的研究较少。LLLT的有效照射剂量和照射方式尚无统一标准,现仍有一些负面报道认为它对骨组织愈合没有作用,因此还不是一个被广泛接受的治疗手段。本研究尝试将LLLT应用于口腔种植领域,以验证LLLT是否能够有效的促进成骨细胞增殖、分化和骨形成,并探讨不同照射方式的LLLT是否有区别,为其应用于临床提供理论依据。本研究采用MTT、流式细胞术、细胞化学钙钴法检测人成骨样细胞MG-63的增殖、细胞周期、细胞凋亡和碱性磷酸酶(alkaline phosphatase,ALP)的表达,观察了LLLT对MG-63生物学特性的影响。采用免疫组化、RT-PCR和Western Blot的方法检测骨桥蛋白(osteopontin,OPN)、I型胶原(collagen I,coll I)的mRNA的表达量和蛋白表达量,观察了LLLT对MG-63细胞外基质蛋白的影响。此外,本研究观察了LLLT对大鼠股骨实验性骨缺损的刺激作用。结果表明,3.75 J·cm-2照射剂量的LLLT可以促进体外人成骨样细胞MG-63的增殖,改变细胞周期时相,抑制细胞凋亡,促进MG-63分泌成骨细胞早期标志物ALP,促进细胞外基质蛋白OPN、coll I的表达,30 J·cm-2照射剂量的LLLT可以促进大鼠股骨实验性骨缺损的骨愈合。此外,实验组中多次照射和单次照射比较并无不同。综上所述,本研究证实了LLLT可以促进成骨细胞增殖、分化,促进动物模型的骨形成,LLLT可能对种植术后的骨愈合有促进作用,具有临床意义,为LLLT应用于口腔种植领域打下坚实的实验基础。更多还原

【Abstract】 As a new light source, a laser possesses many excellent physical characteristics such as tiny divergence angle, brightness, monochromaticity and coherence. The era of laser medicine came when the American scientist Maiman invented the first laser in 1960. Lasers have played an important role in medical diagnosis and therapy. According to the power of the laser utilized, there are two areas of laser therapy: one is for high power lasers which can be used in ablation, cutting, solidification and hemostasis; the other is for low power lasers which can be used for irradiating human tissue, without causing damage, and then promoting its healing.Low level laser(LLL),also called low intensity/power laser, cold laser, soft laser, weak laser and so on, is one kind of visible light and infrared light, which possesses satisfactory monochromatism, between 600~950nm of the wavelength, below 250mW of the output power. At the same time, instead of heat effect, laser has biochemical effects which could not result in irreversible damage to bio-tissue, but rather a similar effect obtained by physical factors such as ultrasound, apuncture and moxibustion. That is called“biostimulation”, and now“photo- biomodulation”.The corresponding therapy is called low-level-laser-therapy (LLLT).For medical workers, there are three areas where LLLT could play an important role: 1) triggering immunological chain reaction, promoting wound healing and tissue repair; 2) reduction of inflammatory process and its associated pain and edema; 3) relief of neurogenic pain influence. Many in vitro and in vivo studies have offered theoretical founation for the clinical application of LLLT. Bone defect is common in oral implantology, which can influence the bone mass and therapeutic schemes. There are several ways of bone repair, for instance, bone graft which includs autogenous bone graft, allogenic bone graft,artificial bone graft, physical stimulus which includes electromagnetic stimulation, recompression therapy, ultrasound. LLLT is a physical treatment which has been demonstrated to improve the proliferation and differen- tiation of osteoblast and bone healing in animal models.In spite of many convicing study of the positive effects of LLLT on bone healing, there are several studies confirmed that LLLT has no effect on osteoblast and bone healing. LLLT is not a widely accepted therapy. Therefore, more experiments are needed to verify the effective dose and irradiation method of LLLT. Meanwhile, few studies were applied to oral implantology. No research is found in domestic area. If LLLT can be applied to bone healing in oral implatology, LLLT may help improve the success ratio of implant.In this study, the effect of LLLT on proliferation,differentiation of MG-63 and bone healing in an animal model was detected. The different methods of LLLT was investigated too. This study can provide a theoretical foundation for the application of LLLT in oral implantology.1 Effect of LLLT on biological characteristics of MG-63OBJECTIVE: To elavuate the biological characteriscs of human osteoblast-like cell MG-63 which was used as an osteoblastic model.METHOD: MG-63 was seeded into H-DMEM with 10% fetal bovine serum. There were 3 groups randomly divided: laser group 1 (L1), laser group 2 (L2), control group(C). The laser was 808 nm, outpower 66 mW, power density 33 mW·cm-2. L1 was multi-irradiated group which was irradiated 3 consecutive days for 40 s per day, that was 1.25 J·cm-2 per day. L2 was single-irradiated group which was irradiated for 120 s on the 1st day, that was 3.75 J·cm-2 and was not irradiated on 2nd and 3rd days.The total dose of both of laser groups was 3.75 J·cm-2 . C was not irradiated. The proliferation, cell cycle, apoptosis, ALP of MG63 were measured by MTT, flow cytometry(FCM) and cytochemical calcium-cobalt method.RESULTS: On the 4th day after single irradiation, MTT of laser groups was higher than that of control group(P<0.05). Compared to control group, laser groups presented more S-stage cells, less G1-stage cells and higher proliferation index(PI,PI=S + G2/ G1 +S + G2)(P<0.05). The apoptosis rates in laser groups were lower than that in control group with significant difference (P<0.05). The expression of ALP in laser groups were higher than that in control group (P<0.05). However, for these 4 indexes, there was no significant difference between laser group 1 and laser group 2 (P>0.05).2 Effect of LLLT on extracellular matrix protein of MG-63OBJECTIVE: To investigated the effect of LLLT on mRNA and protein expression of OPN,coll I of MG-63, and to detect the effect of LLLT on ECMP and the mechanism of LLLT.METHOD: MG-63 was seeded into H-DMEM with 10% fetal bovine serum. There were 3 groups randomly designed: laser group 1(L1), laser group 2(L2), control group(C). The laser was 808 nm, outpower 66 mW, power density 33 mW·cm-2 . L1 was multi-irradiated group which was irradiated 3 consecutive days for 40s per day, that was 1.25 J·cm-2 per day. L2 was single-irradiated group which was irradiated for 120s on the 1st day, that was 3.75 J·cm-2 and was not irradiated on the 2nd and 3rd days.The total dose of both of laser groups was 3.75 J·cm-2. C was not irradiated. The mRNA and protein expression of OPN,coll I of MG63 were measured by immunohistochemical method , RT-PCR and Western Blot.RESULTS: On the 4th day after single irradiation, with immunohistochemical method, the expression of OPN,coll I in laser groups were higher than that in control group (P<0.05). With RT-PCR method, the expression of OPN,coll I in laser groups were higher than that in control group (P<0.05). With western blot, the expression of OPN,coll I in laser groups were higher than that in control group (P<0.05).However, for these 3 indexes, there was no significant difference between laser group 1 and laser group 2 (P>0.05).3 Stimulatory effect of LLLT on bone defect created surgically in rat femurOBJECTIVE:To observe the stimulatory effect of LLLT on bone defect sites created surgically in rat femur, and to evaluate the histological changes including inflammation and bone formation around bone defect.METHOD: Thirty rabbits were randomly divided into 3 groups: laser group 1, laser gourp 2 and control group. Each group was divided into 2 sub-groups according to different sacrificed time: 15d sub-group and 30d sub-group. The laser was 808nm, output 2.6 mW,power density 83 mW cm-2 .L1 was multi-irradiated group which was irradiated 3 consecutive days for 2 min per day, that was 10 J·cm-2 per day. L2 was single-irradiated group which was irradiated for 6 min on the 1st day, that was 30 J·cm-2 and was not irradiated on the 2nd and 3rd days.The total dose of both of laser groups was 30 J·cm-2, C was not irradiated. After an general anesthesia, the femur was perforated with a surgical bone drill 2 mm diameter×1 mm depth. Wound closure was done in layers. The rabbits were killed on 15 d and 30 d after the surgery. The femur was removed, then fixed in 4% paraformaldehyde, decalcified, dehydrated, embedded in paraffin and sectioned for HE staining. The inflammation and bone formation of each group was analyzed under a light micorscope.RESULTS: Control group: At 15 days, the presence of disorganized connective tissue with moderate or severe inflammation. was observed. At 30 days, the wounds were partially filled by newly formed bone tissue composed of numerous osteoblasts differentiating into osteocytes with mild inflammation. Laser group: the presence of organized connective tissue with mild inflammation. was observed. At 30 days, no inflammation was observed. All specimens presented new bone formation and had the defect almost completely filled with bone tissue with characteristics of secondary bone.A large number of blood capillaries were clearly observed in this period. The inflammation score of laser groups were lower than that of control group with significant difference (P<0.05), meanwhile, the bone formation score of laser groups were higher than that of control group with significant difference (P<0.05). However, there was no significant difference between laser group 1 and laser group 2 (P>0.05).In conclusion, LLLT with proper dose may have a positive effect on in vitro or in vivo experiments on early stage, for instance, promoting proliferation and differentiation, improving bone healing of bone defect created in animal model. In this study, there was no different between multi-irradiated group and single-irradiated group. More research on the difference of their biological effect is needed in future.更多还原