| Photodynamic Therapy (PDT) is a kind of photochemistry method for tumor killing. The principle it works for tumor therapy is that the resident photosensitizer in tumor tissue is activated by certain wavelength laser and takes action on oxygen in tumor tissue. Through that, singlet oxygen and freeradicals with high chemical activity are generated, which could react with biology giantmolecule and lead to the destroying of the structures and functions of tumor cells. PDT has been a focus in 1980s. However, because of the immature manufacture technic for photosensitizer and the unstable power output of the laser generator beside its large cubage, the development of PDT was taken no further and the clinical application wasn't get broad. Along with the rapid development of science technology, especially with the invention of semiconductor laser generator and the development of the new type photosensitizer, PDT is attached importance to tumor therapy increasingly in late years. Comparing with operation, chemotherapy and radiotherapy, PDT has the advantages over: 1) minimally invasive; 2) little toxicity; 3) high selectivity; 4) wide applicability; 5) well repeatability; 6) could be used as pamper treatment; 7) could cure precancerous lesion; and 8) maintain features and important organ function. PDT has become one of the routine treatment methods for tumor therapy gradually. Now PDT has got broad clinical application in America, Japan, Canada and England. Clinical practice has proved that PDT can not only cure early cancer and precancerous lesion, but also can get good appeasement effects in late stage cancerpatients, though the disadvantages such as light hypersusceptibility and its ineffective on far inner tumor are still remained.The routine procedure in our clinical work of PDT is as follow. The first laser radiation was taken out 48 hours later after photosensitizer injection. Then the tumor necrosis tissues were cleared away at 96-120 hours after photosensitizer injection. And the second laser radiation was taken out subsequently. We found that the interval time from the first radiation to the second radiation, i.e. 48-72 hours, was too long to endure for patients. They feel discomfort and pains induced by PDT and had got heavy psychology burden, which influenced the patients' rehabilitation severely.Whether the interval time could be shortened while the PDT curative effect maintained? If so, pains will be low down and the hospitalization time will be shortened. And both the economic and psychology burden of the patients will be lightened. In this study, an animal experiment of PDT was taken out for the radiation interval time shortening research. Clinical trial was taken out to explore the best PDT repeat radiation interval time after photosensitizer injection as well.The first part: Study on PDT radiation interval time after photosensitizer injection by animal experimentMethods 4 weeks old KM mice were planted with mouse fibrosarcoma (SI80). The photosensitizer was PHOTOFRIN, and the generator was DIOMED 630 semiconductor laser generator. The PDT groups, which were given PHOTOFRIN (lmg/ml, lOmg/kg, i.v.) and laser radiation (120J/cm2), were devided into 9 test groups according to the different radiation interval time after photosensitizer injection as Tl (lmin), T2 (5 min), T3 (15 min), T4 (30 min), T5 (60 min), T6 (6h), T7 (12h), T8 (24h), and T9 (48h), respectively. Specimens were obtained from the mice killed at once or 48h after radiation, from each experiment subgroup, respectively. We assured that 12 mice in each group were observed for long survive study. All of the left mice were killed for specimens obtaining on the day 40 after radiation. The control group was not given PHOTOFRIN and laser radiation. The characteristic of the shape, color, size, texture, cross-section of the tumor and the skin was observedwhen the mouse was killed. And the specimens were taken for routine pathology examination and electron microscope examination as soon as possible. Contents The observation of the appearance of local radiation region showed that there were no distinguishable characters between all of the experiment groups when the mice were killed at once after radiation, except the degree of skin swell and congestion, which were lightened gradually from Tl to T9. Compare with the groups killed at once after radiation, the skin changed wine color, lost flexibility, stiffened, and had a clear borderline with normal skin in the groups killed at 48h. The eminence tumor under skin seemed flatness, and had more effusion after carving the skin. The effusion was severer in T1-T5 than that in T6-T9. The reason maybe that the amount of photosensitizer in the blood was much higher during that period (T1-T5) , according the principle of pharmicmetabology. So the main factor was blood vessels damaged during that time. But the skins of the groups could be repaired after 40d for the skin was not damaged deathly. Scars were found in some cured mice, and hair growth was difficult. But this phenomena was infrequency in T1-T5. The reason might be that the amount of photosensitizer in tumor and skin tissue at lh and later was much higher, which leads to the severe damage of skin and hair flooicle by radiation. Part of the experiment mice had limb paralysis of the affected side and the paralysis could recover normal after 10-15 days. Only one or two mice got paralysis forever. The highest lower limb paralysis incidence was 33.3% in Tl and the lowest was 8.3% in T9, and the incidence was reduced from T1-T9 gradually. One possible reason is the edema caused by the photosensitizer with high concentration in periphery tissue of tumor could affect the nerves of the affected limb. The other is the singlet oxygen generated and laser radiation could act with the nerves directly. The vascular damage of the nerve tissue could also be included. Since the paralysis was reversibility, the tissue edema might be the main reason for nerve paralysis. The mice death rate due to tumor relapse in the experiment group was about 6.5%, and happened in T1T2T4 and T8T9 groups. Because of the low concentration of photosensitizer in the tumor tissue at 0-30min, there was no enough singlet oxygen and the tumor cell killing is mainly cause by the blood vessels damage. One or twotumor cells might exist and relapse metastasis. Meanwhile, the concentration of photosensitizer in the tumor tissue was also reduced comparatively at 24-48h (T8 and T9), and few tumor cells might evade the PDT's action and lead to relapse. Thus, the best chance to perform the tumor radiation was at l-12h after photosensitizer injection.Most of the mice skins recovered normal on 40 days after PDT in T1-T5. Only some mice skin had cuticular layer incrassation and hair flooicle reduction. In T6-T9, large amount of new connective tissue were seen and found scar formation in some mice skin. It indicated that the best time to perform the tumor radiation was at 0-6h after photosensitizer injection for reducing skin scar formation. Under microscope the tumor cells necrosis were not apparent and there was no difference between experimental groups in the specimens of mice killed at once after irradiation. By electron microscope, we found that the cytochondriomes tumescence was severer and the distance between the cells was increased in T5, T6, and T7. The reason might be higher photosensitizer concentration in the tumor tissue during the time generating more singlet oxygen and active freeradicals, which leads to damage the tumor cells more severely. Tumor specimens got from mice killed at 48h were found tumor cells solidifiability necrosis in extensive region, with seldom survival tumor cells under microscope. And there were no difference between the experiment groups. Under electron microscope, necrosis of the tumor cells was very apparent, intact cell organelle was rarely found. The cytomembrane and karyotheca were broken. Karyoplasms and cytoplast were adulterated. Pycnosis and fragmentation could be found everywhere. The tumor cells were disintegration. There was no difference between the experimental groups. From our observation the tumor cells necrosis belonged to necrosis. The tumor tissue near the center of the substantial mass could be seen typical cell apoptosis as caryotin margin like crescent nucleus and cytoplast vacuolization. The phenomena was as the same as happened in control tumor tissue. Probably this was related to the period of time we took the specimens at once and 48h after irradiation. All results of the observation of microscope and electron microscope supported the PDT machnism were local cytotoxic singlet oxygen action and thedamage of tumor microvasculature. The two factors existed simultaneously, but only one of them was not enough for PDT. It was predominance to do irradiation at 12h after photosensitizer's injection, because of the highest concentration of the photosensitizer in tumor tissue, high PDT efficancy and little influence on normal tissue. The concentration of the photosensitizer in plasm will be raised apparently and the PDT efficancy be increased further if the mouse is given suitable photosensitizer again 30min before irradiation at that time. But this should be confirmed by further animal experiments.Conclusions Skin reaction was gently from the appearance of the skin after irradiation and the paralysis incidence was lower in T6-T9. But some of the cured T6-T9 mice had skin scar formation. The best time to reduce tumor relapse was irradiated at l-12h after photosensitizer's injection. Under electron microscope, the cytochondriomes tumescence was severe in T5T6T7. And there was no difference between the experiment groups in tumor cells necrosis under electron microscope and microscope. As a result, we considered the best irradiation time was at 12h after photosensitizer's injection. In PDT machnism, local cytotoxic singlet oxygen causes the destruction of tumor cells directly and the damage of tumor microvasculature indirectly exist simultaneously. The main action is the damage of tumor microvasculature at lmin-lh after photosensitizer's injection, while local singlet oxygen action as the main factor at 6h-48h after photosensitizer's injection. It was predominance to do irradiation at 12h after photosensitizer's injection, because of the highest concentration of the photosensitizer in tumor tissue, high PDT efficancy and little influence on normal tissue. The concentration of the photosensitizer in plasm will be raised apparently and the PDT efficancy be increased further if the mouse is given suitable photosensitizer again 30min before irradiation at that time. But this should be confirmed by further animal experiments.The second part: Clinical study on PDT irradiation interval time afterphotosensitizer's injectionMethods: 87 patients (50 males and 37 females) either with esophagus cancer (43cases), bronchus cancer (9 cases), nasopharyngeal cancer (20 cases) or laryngeal carcinoma (15 cases) diagnosed by biopsy under endoscope and confirmed by pathologically were brought into the trial, in which there were 7 cases on early stage and 80 cases at the late stage. Patients were divided into two groups at random: 42 cases in routine treatment group and 45 cases in clinical trial group. The first radiation time in the routine treatment group was at 48h after photo frin injection and the second at 96h. The first radiation time in the clinical trial group was the same as the routine treatment group while the second at 72h. All of the patients received photofrin (2.5mg/ml) by intravenous administration at the dose of 2 mg/kg b.w. in 5% glucose.The patients should be avoided sunlight strictly to prevent photoallergy for 4-6 weeks. The radiation was performed with 630 nm light (DIOMED) delivered through cylinder diffusing tip quartz fibers passed through the biopsy channel of endoscope. The curative effect was checked by endoscopy 1-2 months after PDT. The lumen obstruction, karnofsy score and weight change were also evaluated. Contents The early reactions to the first irradiation of two groups were no difference at once after irradiation. The main response was severe edema in tumor tissues, especially in the outer coating of the mass. The tumor tissue changed fragile. 45min after the first radiation, the part of the tumor tissues radiated directly became brown and shrink from edema and paleness. When the second radiation was performed, the volume of the tumor tissue shrank a lot in the routine treatment group. The color was dark brown. And the necrosis tumor tissue was just like jelly. There was little oozing of blood after the clearance of the necrosis tissue. In the clinical trial group, the volume of the mass also shrank, the color turn wine, the outside necrosis tissue changed white, and the necrosis fragments were easy to peel off, the new granulation tissue or the deeper layer tumor tissue came out with no bleeding. There was no difference between the two groups under endoscopy. The results showed that the CR+SR rate were 88.1% in routine treatment group and 88.9% in cilinical trial group after the evaluation of the effectiveness 1-2 months after PDT. The curative effect was quite well in the near future, and the lumen obstruction was improved in evidence. The lumen obstruction rate was reduced from 50%(21/42) to 9.5%(2/42) in routinetreatment group, and from 40%( 18/45) to 8.9%(4/45) in clinical trial group. The remission rate of lumen obstruction was 81.0% and 83.3% respectively. Karnofsky score and weight amelioration rate were 64.3% and 59.5% respectively after PDT in routine treatment group compared with clinical trial group as 68.9% and 68.9%. All the evidences showed that there was no difference in lumen obstruction, karnofsy score and weight change between the two groups. And there was no difference in complications and side-effects between the groups, as well as the complications and side-effects..Conclusion There was no difference between the two groups in the appearance configuration of the tumor at early stage, repeated irradiation and 1 month after PDT. The CR+SR rate were 88.1% in routine treatment group and 88.9% in cilinical trial group with no difference too. The lumen obstruction were improved in evidence. And there were no difference in the remission rates of lumen obstruction, the amelioration rates of Karnofsky score and weight change, the incidence of complications and side-effects between the two groups. As a result we considered that shortened the PDT repeat irradiation interval time after photosensitizer's injection could not affected clinical cured effect of PDT. On the contrary, the hospitalization time of the PDT patients could be shortened. Psychologyly, this could reduced the mental load of the patients, make the patients received PDT easier and in favor of recuperating patients' health.
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