阅读说明：本技术 锡中卟啉在制备地中海贫血治疗药物中的应用 (Application of tin mesoporphyrin in preparation of medicine for treating thalassemia ) 是由 陈慧勇 刘静 彭元亮 张海航 于 2021-01-18 设计创作，主要内容包括：本发明公开了锡中卟啉在制备地中海贫血治疗药物中的应用,本发明使用锡中卟啉腹腔注射β地中海贫血小鼠,发现锡中卟啉可以让地贫小鼠无效红系生成回归正常,有效缓解红细胞的α/β珠蛋白链不平衡,提升红细胞寿命、红细胞总数及血红蛋白水平,同时降低血清及肝脏的铁含量及脾肿大。经锡中卟啉治疗后,地贫小鼠无效造血、贫血和铁超载均得到有效缓解,无明显毒副作用,治疗实施方便,高剂量之后,只须低剂量维持即可保持疗效。(The invention discloses application of stannum mesoporphyrin in preparation of a medicine for treating thalassemia, wherein the stannum mesoporphyrin is used for intraperitoneal injection of a beta thalassemia mouse, so that the stannum mesoporphyrin can enable ineffective erythroid generation of the thalassemia mouse to return to normal, the unbalance of alpha/beta globin chains of erythrocytes is effectively relieved, the service life of the erythrocytes, the total number of the erythrocytes and the hemoglobin level are prolonged, and the iron content of serum and liver and splenomegaly are reduced. After the treatment of tin mesoporphyrin, the ineffective hematopoiesis, the anemia and the iron overload of the thalassemia mice are effectively relieved, no obvious toxic or side effect is generated, the treatment and implementation are convenient, and after the high dose is adopted, the curative effect can be kept only by maintaining the low dose.)

1. Application of tin mesoporphyrin in preparing medicine for treating thalassemia is provided.

2. A medicine for treating thalassemia is characterized in that the effective component of the medicine is tin mesoporphyrin.

Technical Field

The invention relates to a new application of tin mesoporphyrin, in particular to an application of tin mesoporphyrin in preparing a medicine for treating thalassemia. Belongs to the technical field of medicine.

Background

Thalassemia (abbreviated as "thalassemia") is one of the most common genetic diseases of hemoglobin worldwide. The main pathogenic mechanism is that the mutation of alpha or beta globin gene inhibits the synthesis of globin subunit, so that the alpha/beta globin chain in erythrocyte is unbalanced, resulting in deposition of globin in erythrocyte membrane, unstable and easy-to-break structure of erythrocyte membrane, and the pathological features of ineffective hematopoiesis, anemia, iron overload, etc.

The treatment means of the anemic patients at present mainly comprise blood transfusion and iron removal combined treatment, spleen resection treatment, bone marrow transplantation treatment and gene therapy. The treatment of blood transfusion and iron removal needs to be carried out for a long time, and the treatment cannot be cured and is expensive, so that serious economic burden is caused to families of patients; spleen resection treatment only has good curative effect on the intermediate thalassemia patients, reduces the autoimmunity of the patients by splenectomy, and improves the risk of diseases; bone marrow transplantation therapy, which is a mature technology for only curing the thalassemia patients, but has the disadvantages of excessively high treatment cost, little and difficult bone marrow matching and incapability of treating the thalassemia patients in a large range; gene therapy, the technology is not fully mature, and the research reports that the treatment cost is up to millions of dollars, which is cost that most families cannot bear and the treatment cannot be generally carried out. The invention discovers a novel strategy for treating the thalassemia by research, targets the thalassemia and has no effective hematopoietic root, can effectively improve the anemia, relieve secondary iron overload blood, reduce possible complications, reduce the family economic burden of patients and increase the treatment universality of the patients.

Based on the defects of the current methods for treating thalassemia patients, a strategy which is more economical and reliable and can be generally treated is found to be a new direction for treating thalassemia.

Tin mesoporphyrin has been reported to be used for treating infant jaundice, and no reports on the use of tin mesoporphyrin for treating thalassemia exist at present.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides the application of tin mesoporphyrin in the preparation of a medicine for treating thalassemia.

In order to achieve the purpose, the invention adopts the following technical scheme:

1. application of stannum mesoporphyrin (SnMP) in preparing medicine for treating thalassemia is provided.

Preferably, the dose of the SnMP treatment in the thalassemia mice is 25 mu mol/kg, and the SnMP treatment is performed by intraperitoneal injection for 3 times per week and is maintained for 1 time per week after 4 weeks of continuous administration.

2. A medicine for treating thalassemia contains tin mesoporphyrin as effective component.

The invention has the beneficial effects that:

iron overload is one of the remarkable features of thalassemia patients, and is accompanied by various complications, such as growth and development inhibition, diabetes, liver cancer and the like. The iron cycle of the body involves several processes of absorption, transport, utilization/storage and recycling of iron. According to the invention, the tin mesoporphyrin is used for intraperitoneal injection of the beta thalassemia mouse, and the finding that the tin mesoporphyrin can enable the ineffective erythroid generation of the thalassemia mouse to return to normal, effectively relieves the unbalance of alpha/beta globin chains of erythrocytes, improves the service life of the erythrocytes, the total number of the erythrocytes and the hemoglobin level, and simultaneously reduces the iron content of serum and liver and splenomegaly.

After the treatment of tin mesoporphyrin, the ineffective hematopoiesis, the anemia and the iron overload of the thalassemia mice are effectively relieved, no obvious toxic or side effect is generated, the treatment and implementation are convenient, and after the high dose is adopted, the curative effect can be kept only by maintaining the low dose.

Drawings

FIG. 1 shows the effect of SnMP on the deposition of alpha globin in peripheral red blood cells of mice detected by using Triton-acetic acid-urea (TAU) gel electrophoresis technology: a is TAU glue electrophoresis staining result, B is normalized alpha globin deposition electrophoresis band gray value statistics. ns has no significant difference; 0.0001.

FIG. 2 is a graph showing the effect of SnMP on the half-life of peripheral red blood cells of mice detected by flow cytometry. ns has no significant difference; p < 0.05; 0.001.

FIG. 3 shows the effect of SnMP on the peripheral blood anemia index of mice according to the present invention: a is the number of erythrocytes, B is the hemoglobin concentration, and C is the number of reticulocytes. ns has no significant difference; p < 0.01; 0.0001.

Figure 4 is a graph of the effect of SnMP treatment on mouse spleen: a is spleen appearance, B is relative spleen weight/body weight ratio statistics. ns has no significant difference; 0.0001.

FIG. 5 is a graph showing the effect of SnMP on the serum iron concentration of mice detected by a microplate reader. P < 0.05.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and examples, which are provided for the purpose of illustration only and are not intended to limit the scope of the invention.

Example 1: in the embodiment, the influence of SnMP on the alpha globin deposition in the peripheral red blood cells of the mice is detected by adopting a TAU gel electrophoresis technology

The experimental steps are as follows:

1. age and sex matched wild type (purchased from sleek scapand, sho, han) and β -thalassemia mice (purchased from jackson laboratories, for model construction see reference) were each divided into 2 groups: one group was intraperitoneally injected with 200. mu.L of Phosphate Buffered Saline (PBS) of pH7.2-7.4 as a control group, and one group was intraperitoneally injected with 200. mu.L of SnMP as an experimental group. The dose of SnMP treatment is 25 mu mol/kg, 3 times per week of intraperitoneal injection, and after 4 weeks of continuous injection, 1 time per week of maintenance.

2. Preparing a red blood cell membrane sample;

1) using an anticoagulation centrifuge tube to collect 4 mu L of mouse blood;

2) the blood cells were centrifuged and washed 1 time with pre-cooled PBS (1mL, 2000rpm, 5 min, 4 ℃) (8. mu.L of the supernatant was taken and 10. mu.L of TAU loading buffer was finally added as a standard for electrophoresis);

3) adding 200 μ L of 0.1 × PBS, placing on ice, and lysing for 50 min (or repeatedly freezing and thawing at-80 deg.C for 2 times);

4) discarding the supernatant, washing the precipitate with 0.1 × PBS 2 times at the highest speed for 5 min at 4 deg.C;

5) the pellet was dissolved with 10. mu.L of TAU loading buffer.

3. TAU gel electrophoresis, elution and development, and photographing.

The experimental results are shown in fig. 1 (wherein, a is the result of electrophoretic staining with TAU gel, and B is the gray value statistics of the normalized alpha globin deposition electrophoretic bands of each group): after 4-6 weeks of SnMP treatment, peripheral red blood cell alpha globin deposition in thalassemia mice was significantly reduced compared to PBS control, while wild type mice had no significant change. The SnMP can be used for specifically improving the phenomenon that the peripheral blood alpha/beta globin chain of the poor mice is unbalanced.

Example 2: in the embodiment, the influence of SnMP on the half-life of peripheral red blood cells of mice after biotin labeling is detected by flow cytometry

The experimental steps are as follows:

1. mice were grouped and treated as in example 1;

2. collecting 200 mu L of peripheral blood of mice in a PBS/SnMP treatment group by an anticoagulation centrifugal tube;

3. adding 300 mu M biotin labeling solution, mixing evenly and lightly, and incubating for 60 minutes at 37 ℃ in a dark place;

4. washing the cells for 1 time after the marking is finished, and injecting the marked red blood cells into a wild type receptor mouse through orbital vein;

5. taking 24 hours in a cell transfusion receptor mouse as a starting point, taking 2-3 mu L of peripheral blood every 7 days, adding Phycoerythrin (PE) coupled streptavidin, incubating for half an hour in the dark, detecting the proportion of PE positive cells by an up-flow type instrument, analyzing the result and carrying out statistical analysis.

The experimental results are shown in fig. 2: the red blood cell decay of the thalassemia mouse-SnMP treated group was significantly slowed compared to the PBS treated group, whereas there was no significant difference between the SnMP and PBS treated groups of wild type mice. Indicating that SnMP treatment can effectively increase and prolong the half-life of red blood cells in poor mice.

Example 3: in the embodiment, the influence of SnMP on the peripheral blood anemia index of the mouse is detected by adopting a blood routine method

1. Mice were grouped and treated as in example 1;

2. collecting peripheral blood of the mouse after PBS/SnMP treatment for 2-6 weeks by an anticoagulation centrifugal tube;

3. peripheral blood was diluted with a 6-fold volume of blood cell analysis diluent, and the peripheral blood was loaded into a blood analyzer (Sysmex, Japan) and the data was read.

The experimental results are shown in fig. 3: after the SnMP is treated for 4-6 weeks, the peripheral blood anemia indexes of thalassemia mice, namely the number of red blood cells (A in figure 3) and the hemoglobin level (B in figure 3), are obviously recovered; in addition, the number of peripheral blood reticulocytes in the thalassemia mice decreased significantly from 2 weeks after SnMP treatment (C in fig. 3) and decreased with increasing treatment time. Therefore, the SnMP treatment can effectively improve the peripheral blood anemia index of the thalassemia mice.

Example 4: this example demonstrates the effect of SnMP treatment on mouse spleen

The experimental steps are as follows:

1. mice were grouped and treated as in example 1;

2. weighing and recording the mice, dissecting the mice, taking out the complete spleen, weighing and recording the spleen;

3. the spleens of the mice were arranged on a background clear paper, photographed and data processed, statistically analyzed, and the spleen body mass index was calculated.

The results of the experiment are shown in FIG. 4. The result shows that the appearance (A in figure 4) and the weight (B in figure 4) of the spleen of the thalassemia mouse are obviously improved after 4-6 weeks of treatment by SnMP, which indicates that the SnMP can effectively relieve the splenomegaly of the thalassemia mouse.

Example 5: in the embodiment, the influence of SnMP on mouse serum iron is detected by adopting a microplate reader

The experimental steps are as follows:

1. mice were grouped and treated as in example 1;

2. collecting the serum of the mouse treated by PBS/SnMP by using a common centrifugal tube;

3. performing related treatment on mouse serum by a serum iron kit (purchased from Nanjing institute of bioengineering), performing centrifugation for 10 minutes at 3500 rpm in boiling water bath for 5 minutes, and collecting supernatant;

4. and detecting the light absorption value of the supernatant at 520nm by using a microplate reader, performing statistical analysis, and calculating the mouse serum iron.

The results of the experiment are shown in FIG. 5. The result shows that the serum iron concentration of the thalassemia mouse is remarkably reduced after the SnMP treatment for 4-6 weeks, which shows that the SnMP can effectively relieve the iron overload phenomenon of the thalassemia mouse.

Although the embodiments of the present invention have been described with reference to the accompanying drawings, the scope of the present invention is not limited thereto, and various modifications and variations which do not require inventive efforts and which are made by those skilled in the art are within the scope of the present invention.