| Objectives:On the basis of previous work,we diagnosed the gene of thalassemia and Glucose-6-Phosphate Dehydrogenase deficiency(G6PD deficiency)in Dai population of Yunnan Province,and found out whether there is a difference between the infection of defective red blood cells and normal red blood cells by Plasmodium falciparum infecting the red blood cells of people with these two diseases and normal people,so as to understand the relationship between malaria prevalence and the above diseases.To understand the cellular biological mechanism of the antimalarial effects of thalassemia and G6 PD deficiency.Methods:1.Whole blood samples were collected from Dai people in Nongzhang Town,Yingjiang County,Dehong Prefecture,Yunnan Province;2.Genetic diagnosis of thalassaemia,enzyme activity detection of G6 PD deficiency,and gene mutation site analysis of whole blood samples;3.Use the P.falciparum strain 3D7 to infect G6 PD deficiency,thalassaemia disease and normal red blood cells.4.Use optical microscope,analytical flow cytometer,chemiluminescence instrument,enzyme-linked immunodetector to observe the morphology,parasitemia and determination of malaria pigment content of malaria parasites cultured in vitro.5.Apply electron microscopy technology to conduct in-depth analysis of the internal structural changes of red blood cells and malaria parasites during the culture process to understand their changes in the microscopic field of view.Results:1.Genetic diagnosis of thalassemia:There were 28 cases of thalassaemia in the52 samples.Among them,α-thalassaemia accounted for the highest proportion,and αcompound β thalassaemia accounted for the least,and there was a significant difference between the two(P < 0.05,Fisher’s exact test).Genotype and composition ratio of G6 PD deficiency results: In this sample,there were 29 patients with G6 PD deficiency,the proportion of women was higher than that of men,and there were significant differences in statistical analysis(P <0.0001,Fisher’s exact test).The proportion of G6 PD gene mutation sites was 1311 C >T,which was statistically different from that of 392 G > T(P < 0.01,Fisher’s exact test).2.Light microscope resultsMicroscopic observation of sample blood smears:1)Thalassemia,G6 PD deficiency,and patients with both diseases: their erythrocytes were observed to have a crumpled,malformed cell shape after 96 hours of in vitro culture of P.falciparum,and most of them showed cell crumpling and malformation;when the developmental stage of the intraerythrocytic phase of the life history of P.falciparum was observed,P.falciparum showed retarded development and difficulties in invading and parasitizing erythrocytes,resulting in unhealthy cytoplasmic dispersion of the worms2)Normal erythrocytes: observe the shape of erythrocytes,the morphology is normal,and there is no disappearance of erythrocyte light stained area or other special morphology;observe the developmental stage of intraerythrocytic stage of Plasmodium life history,Plasmodium development period is more consistent,and the worm body is in good condition.The statistical analysis of the parasite density: The parasite density of cultured P.falciparum from the erythrocytes of the healthy persons was significantly higher than that from the erythrocytes of the patients with thalassemia,G6 PD deficiency and both diseases(P < 0.05,One Way ANOVA).However,there was no statistically significant difference in protozoal density comparison in defective red blood cell culture P.falciparum worm.4.Plasmodium infection rate result:1)Flow cytometry: There was a statistically significant difference between thalassemia and G6 PD deficiency and plasmodium infection in red blood cells(P <0.05,One Way ANOVA).Compared with patients with thalassemia,G6 PD deficiency and the combination of the two diseases,the infection rate of plasmodium in red blood cells of healthy people was higher,and the statistical difference was significant(P < 0.0001,One Way ANOVA).2)Chemiluminescence meter results: There was no statistical difference in the rate of protozoal infection between red blood cells of patients with thalassemia and G6 PD deficiency and combined with two anemias.Normal people had a higher rate of protozoal infection with red blood cells than patients with thalassemia,G6 PD deficiency,and patients with two diseases,and there was a significant difference in statistical results(P < 0.0001,One Way ANOVA).The above comparison of malaria parasite infection rates proves that normal red blood cells are more susceptible to malaria parasite infection.4.Transmission electron microscopy results: defective erythrocytes(erythrocytes from patients with thalassemia and G6 PD deficiency,combined with both types of anemia)mostly showed significant changes in shape after culture,with bizarre morphology,irregular shape,long tail shape,etc.In addition to the variable appearance,intracytoplasmic vacuoles were common and the cell membrane surface was incomplete,with local or multiple disintegrations.Normal human erythrocytes have a regular shape and uniform size under electron microscopy,with intact cytosol and few openings.Conclusions:1.In the sample of Dai people in Yunnan Province,the carrying rate ofα-thalassaemia was higher than that of β thalastaxaemia;The five common gene mutation sites in G6 PD deficiency were mainly 1311 C > T.2.Defective red blood cells have problems such as red blood cell deformation and protozoa stunting after culture of P.falciparum,while normal red blood cells still maintain normal appearance after culture of P.falciparum,and the development of protozoa is not restricted.3.Normal red blood cells are more susceptible to infection than red blood cells of patients with thalassemia and G6 PD deficiency.4.Under electron microscopy,after the defective red blood cells are cultured,the shape of the red blood cells is changeable,and many large vacuoles appear in the cytoplasm. |
PDF Full Text Request