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Application of photodynamic therapy and equipment and photosensitizer to treating osteosarcoma

A photodynamic therapy and photosensitizer technology, which can be used in phototherapy, medical preparations containing active ingredients, antitumor drugs, etc., and can solve the problems of lack of safe and effective treatment of osteosarcoma.

Inactive Publication Date: 2014-12-10
SHANGHAI TENTH PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] Aiming at the current lack of safe and effective methods for treating osteosarcoma, the present invention provides a new method for treating osteosarcoma, and the application of photodynamic methods, equipment and photosensitizers in treating osteosarcoma

Method used

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  • Application of photodynamic therapy and equipment and photosensitizer to treating osteosarcoma
  • Application of photodynamic therapy and equipment and photosensitizer to treating osteosarcoma
  • Application of photodynamic therapy and equipment and photosensitizer to treating osteosarcoma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1, photosensitizer HMME uptake experiment

[0041] Osteosarcoma cells (LM8) and myoblasts (C2C12) in the logarithmic growth phase were digested, centrifuged and counted at 2×10 per well. 5 A density of 35mm 2 Petri dish. After 24 hours, according to the relevant literature and the previous research basis, HMME (20 μg / ml) was added to incubate for 0, 1, 2, 3, 4, 6, 8, 12 and 24 hours; the two cell lines were incubated in different concentrations of HMME (0, 10, 20, 30, 40 μg / ml) for 4 hours. The cells were digested and centrifuged to suspend in PBS, and the fluorescence intensity of HMME in the cells was detected in the PerCP-Cy5-5-A channel of the flow cytometer.

[0042] Such as Figure 1A with Figure 1B As shown, when the concentration of the photosensitizer HMME is 20 μg / ml, the fluorescence intensity in the cells increases with the extension of the incubation time; when the cells are incubated in different concentrations of the photosensitizer, the f...

Embodiment 2

[0043] Embodiment 2, MTT method detects the optimal incubation time of HMME in sarcoma cells

[0044] Take 5×10 3 LM8 cells per well were seeded in 96-well plates, and after 24 hours, LM8 cells were incubated with HMME for 1 hour, 2 hours, 4 hours, 8 hours, and 12 hours, and the laser energy density was 9 J / cm 2 , HMME concentrations were 10, 20, 30 μg / ml, after 24 hours of light. Add 20 μl MTT to each well and incubate for 4 hours, discard the supernatant and add 150 μl DMSO, incubate in the dark for 10 minutes and shake, and detect OD with a microplate reader 490nm value. Calculate the optimal incubation time of the photosensitizer.

[0045] Survival rate (%)=(experimental group OD 490nm - Zero hole OD 490nm ) / (OD of blank control group 490nm - Zero hole OD 490nm )×100%.

[0046] Such as figure 2Shown, the incubation time is 4h, with 9J / cm 2 (20, 30μg / ml) treated LM-8, compared with 1h (P2 When the concentration of HMME increased, the killing effect was enhanced a...

Embodiment 3

[0047] Embodiment 3, MTT method detects sarcoma cell viability

[0048] Take 5×10 3 Osteosarcoma cells (LM8, MG63, Saos-2), chondrosarcoma cells (SW1353), Ewing sarcoma cells (TC71) and rhabdomyosarcoma cells (RD) were seeded in 96-well plates. 5%CO 2 , 37°C, and a saturated humidity incubator for 24 hours, adding different concentrations of HMME (10, 20, 30 μg / ml) to continue the cultivation. Discard the unbound photosensitizer after 4 hours, wash twice with PBS, add 2% medium, and irradiate with a 630nm laser, and the light energy density is 0, 3, 6, 9 J / cm respectively 2 , the spot diameter is 4cm, and the irradiation time is 2min. Set dark poison group (without light but with photosensitizer), laser group (without photosensitizer but with light), blank group (without photosensitizer and without light). After 24 hours, add 20 μL MTT to each well and incubate for 4 hours, discard the supernatant and add 150 μl DMSO, incubate in the dark for 10 minutes and shake, and dete...

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Abstract

The invention provides a novel method for treating osteosarcoma, and application of photodynamic therapy and equipment and a photosensitizer to treating osteosarcoma, wherein the photosensitizer can be selectively gathered in osteosarcoma cells. Animal in vivo experiments show that PDT taking HMME as the photosensitizer can be used for effectively inhibiting growth of osteosarcoma, and moreover, a wide necrotic zone which is homogenously red-stained at the tumor site is observed by virtue of HE staining; the wide necrotic zone is black and is incrustated in the periphery with a lot of inflammatory cells infiltrated; residual tumor cells have changes in karyopyknosis, vacuolar degeneration and the like. According to the invention, HMME-PDT is verified for the first time that HMME-PDT is mainly used for inducing apoptosis of osteosarcoma cells and is closely related to a caspase dependent apoptosis pathway.

Description

technical field [0001] The invention relates to a method, equipment and photosensitizer for treating osteosarcoma, in particular to a photodynamic therapy and the application of the equipment and photosensitizer in treating osteosarcoma. Background technique [0002] Bone and soft tissue sarcoma (osteosarcoma) accounts for about 1% of all kinds of malignant tumors. Although it is less common than some common tumors such as intestinal cancer, breast cancer, and lung cancer, because such tumors are more likely to occur in adolescents and middle-aged people, the degree of malignancy high. The condition is complex and changeable, the treatment is quite difficult, the prognosis is poor, and the disability and mortality rate is high, which brings great suffering to patients and great harm to society. [0003] In recent years, the diagnosis and treatment of bone and soft tissue tumors have made great progress. Especially since the standardized implementation of neoadjuvant chemot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K41/00A61P35/00A61N5/06
Inventor 蔡郑东曾辉华莹奇周承豪孙梦熊尹飞单连成孙伟王卓莹陈泉池
Owner SHANGHAI TENTH PEOPLES HOSPITAL
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