Introduction
Viscous storage disease type IIAlso known as inclusion cell disease (inclusion cell disease), referred to as I-cell disease. Its clinical features are more like Hurler syndrome, manifested by obvious clinical and X-ray abnormalities at birth, slow response, but no mucopolysaccharide. Skin fibroblasts are cultured with a large number of large cytoplasmic inclusion bodies. Leroy et al. (1967) first discovered the disease, and Spranger (1970) classified it as a mucolipidosis type II.
Cause
(1) Causes of the disease
The cause of viscous storage disease is autosomal recessive inheritance.
(two) pathogenesis
The disease is caused by a variety of hydrolase defects, the use of patient fibroblast mixed culture found that the basic biochemical defect of this disease is the abnormal recognition site of several lysosomal enzymes. The normal combination of lysosomal enzymes requires intracellular synergy, ie, cell synthesis, secretion of hydrolase, cell surface recognition of hydrolase, followed by uptake by lysosomes, and immobilization in lysosomes. Cell surface recognition of hydrolases requires specific sites on both the cell surface and lysosomal hydrolases. At present, it is believed that several lysosomal enzymes are involved in the recognition of the lysosomal enzymes caused by mutations in the type II gene of the viscous storage disease, that is, the recognition subunits in the molecular structure of the enzyme are abnormal.
Strecker et al (1976) found that tissue neuronase deficiency in patients with this disease may be related to abnormalities in the recognition sites of various lysosomal hydrolases, such as hexosaminidase, β-glucuronidase, β- The recognition site of galactosidase and the like is deficient, causing excessive neutral, acidic mucopolysaccharide and mucus deposition in the tissue cells to cause disease.
Pathology: Histological changes are mainly limited to mesenchymal cells. Liver biopsy showed that most Kupffer cells were normal, and the liver cells were filled with various types of inclusion bodies, which contained non-enveloped fat droplets and 0.7 to 50 μm inclusion bodies. These inclusions showed different The degree of rectangular crystallinity is located in the microfilament material, and the microfilament material is surrounded by a film. The most inclusion body in hepatocytes contains a hydrophilic substance which has a layered structure or a small spherical shape. The kidney-packed (Bowan) sac cells showed an inclusion body similar to that of hepatocytes, which was 0.3-4.0 μm in size and positive for PAS staining. Neurons and glial cells in the brain show normal under the microscope, but under the electron microscope, neurons, stellate cells and perivascular epithelial cells have transparent inclusion bodies of 0.3-1.5 μm, surrounded by envelope vacuoles, empty There is a uniform and fine particle matrix in the bubble, which contains a small amount of layered structure and a denser hydrophilic inclusion body surrounded by a coating. The biopsy of the humerus showed that the chondrocyte cytoplasm contained a large amount of lysosomes, and the lysosome contained small, reticular, granular and membrane-containing inclusion bodies. Histochemical staining showed that neutral, acidic mucopolysaccharides and mucus were deposited in the inclusion bodies. Microscopic examination revealed significant ossification of the cartilage, accompanied by complete hyperplasia and hypertrophic cartilage decalcification. The original trabecular bone is short, and the near-bone trunk is mixed with a persistent cartilage area. The fibroblasts of all organs contain a large number of vacuoles surrounding the envelope, so that the fibroblasts are clearly balloon-like. Peripheral lymphocytes can also have most large vacuoles. Inclusion bodies in all organ fibroblasts are pleomorphic, ranging from transparent vacuoles to dense hydrophilic bodies or layers.
symptom
Many abnormalities can be found at birth, such as congenital dislocation of the hip, male inguinal hernia, rough face, abnormal bones, limited movement, and low systemic muscle tone. However, it is generally not possible to make a diagnosis during the neonatal period. About 6 months after birth, the baby's length can be within the normal range, but there is low systemic muscle tension, can not roll in bed, poor head support and many abnormal appearance, some cases may have severe mental retardation.
The face is progressive and rough, the forehead is high, the internal hemorrhoids are suede, the eyelids are thick, the nose is flat, the nose is turned up, and the gums are hyperplasia. The cornea is clear, but a subtle diffuse matrix abnormality can be found by slit lamp examination. Children with increased nasal discharge can recurrent respiratory infections, pneumonia and otitis media. Joint movement is limited and there is contracture. The skin is tight and thick, the abdomen is bulging, and the liver is enlarged. Slow movement. Generally, systolic murmur, short neck, thoracic deformity and microcephaly may occur after one year of age.
According to clinical symptoms, X-ray findings, laboratory findings, and no excessive mucopolysaccharide excretion in the urine, the disease can be initially diagnosed, and the cultured skin fibroblasts and various tissues contain inclusion bodies, and there are many lysosomes. Enzyme defects such as β-galactosidase, N-acetyl-galactosaminease, β-glucosamine, arylthioesterase, fucosidase, etc., and the activity of these enzymes in serum is increased. Electron microscopy showed swelling of the lysosome. It is filled with a dense substance with a coating. You can diagnose.
diagnosis
This type of viscous storage disease needs to be differentiated from other types of viscous storage diseases.
complication
Infants can have congenital dislocation of the hip and male infants can have inguinal hernia. It can also be complicated by liver enlargement. After one year of age, systolic murmur, short neck, thoracic deformity and microcephaly can occur.
treatment
(a) treatment
No special therapy. Severe deformity should be surgical orthopedic. For patients with infection and heart failure, symptomatic treatment should be performed.
(two) prognosis
Poor prognosis, usually within 2 to 8 years of age, more death from infection and heart failure.
prevention
1. First-level prevention of genetic diseases, in addition to the epidemiological survey from the perspective of the entire population, carrier detection, population genetic monitoring and environmental monitoring, marriage and birth guidance, efforts to reduce the incidence of genetic diseases in the population In addition to improving the quality of the population, effective prevention measures must be taken for individuals to avoid the birth of genetically ill offspring (ie, eugenics) and genetic variation. The usual measures include: premarital examination, genetic counseling, prenatal examination and Early treatment of genetic diseases.
(1) Pre-marital examination: pre-marital examination (ie, marriage health care), it is an important link to ensure the happiness of both men and women after marriage and the health of future generations. The pre-marital examination focuses on: 1 investigation of genetic diseases, including detailed inquiries about the health status of men and women and their family members, past medical history and treatment, especially the presence or absence of congenital malformations, genetic history and close relatives. If necessary, family investigation, blood group examination, chromosome examination or genetic diagnosis should be carried out to detect carriers; 2 comprehensive physical examination, mainly for acute infectious diseases, tuberculosis, or severe heart, liver and kidney diseases, chronic urinary tract Inflammation and other diseases that can seriously threaten the health of the individual or spouse, as well as the serious anemia of the woman, diabetes, and other diseases that can affect the fetus, and mobilize the child to be married after being cured; 3 check the male and female reproductive organs, check out Sexual organ malformations, hermaphroditism and other diseases, so that measures can be taken very early.
(2) Genetic counseling: genetic counseling is a case in which the clinician and genetics answer the questions about the etiology, hereditary mode, diagnosis, treatment and prognosis of hereditary diseases. Estimate the probability that the patient's child will suffer from a disease, and provide advice and guidance for the patient and his relatives to refer to. The significance of genetic counseling is: 1 to alleviate the physical and mental pain of patients, reduce the psychological pressure of patients and their relatives, help them to treat genetic diseases correctly, understand the probability of morbidity, take correct prevention and treatment measures; 2 reduce the genetic disease of the population Incidence, reducing the frequency of harmful genes, and reducing transmission opportunities.
2. The general principle in the treatment of genetic diseases is to ban their refusal, to remove the rest, to adjust their metabolic balance, to prevent the appearance of symptoms.
(1) Correcting metabolic disorders: This is the most important method for the treatment of hereditary metabolic diseases. With the deepening of the understanding of the pathogenesis and intermediate processes of hereditary metabolic diseases, the scope of application of this method is also expanding.
1 diet control (forbidden): When the metabolic abnormalities cause the lack of certain essential substances in the body, they are supplemented by diet; when the metabolic substances are stored, the intake of the metabolites or their precursors is restricted. Maintain balance. A low phenylalanine diet in patients with phenylketonuria is a good example. In addition, it can also reduce the intake by limiting the absorption of specific substances. For example, patients taking phenylketonuria take phenylalanine aminohydrolase capsules, which can convert phenylalanine in food into phenylacrylic acid and be eliminated. .
2 reduce the substrate (to the rest): when the disease caused by the metabolism of harmful substances, you can control or improve the disease by reducing harmful substances and reducing the concentration of its precursors and metabolic derivatives, removing or reducing its toxic effects Symptoms. The main methods are: A. chelation or promotion of excretion; B. plasma exchange and affinity binding; C. altered metabolic pathway; D. surgical bypass surgery; E. metabolic inhibition.
3 product substitution (to make up for it): When the important enzymatic reaction product is insufficient and cause disease, the corresponding essential end product can be directly supplemented. For example, to give pituitary dwarf patients growth hormone, hemophilia patients with anti-hemophilia protein (clotting factor), to the corresponding immunoglobulin in patients with hereditary immunodeficiency.
(2) Correcting abnormal enzyme activity:
1 Coenzyme supplement: Some genetic diseases, abnormal enzyme activity may involve:
A. A binding site for a specific coenzyme or vitamin.
B. Active coenzyme transport or biosynthesis processes leading to abnormalities. Many coenzymes are required for the normal activity of whole enzymes. Therefore, supplementing the coenzyme component is also an effective method to induce an increase in enzyme activity, which can slow down the degradation rate of the whole enzyme in the cell, increase the half-life of the enzyme, and reduce the Michaelis constant (Km) of the enzymatic reaction, which has been used. This method treats more than 25 genetic diseases. For example, cobalamin (B12) is used to treat various anemias and methylmalonic aciduria.
2 Enzyme Induction or Feedback Inhibition: Another treatment for enzyme deficiency levels is to use drugs to increase residual enzyme activity to improve metabolic levels. For example, phenobarbital and related drugs can significantly stimulate the formation of smooth endoplasmic reticulum and accelerate the synthesis of specific enzymes in the endoplasmic reticulum, including liver UDP glucuronyltransferase, in the treatment of Gibert syndrome with phenobarbital. And the Crigler-Najjar syndrome provides a theoretical basis.
Feedback inhibition is an important form of many metabolic regulation. For the accumulation of substrates or their precursors caused by certain enzyme defects, feedback inhibition by other bypass metabolism can improve enzyme activity and reduce accumulated substrate. Inhibition has been used as a method of treating acute porphyria.
3 Allogeneic transplantation: by implanting the same kind of cells, tissues or organs containing normal genes into genetically ill individuals, in order to produce corresponding active enzymes and other gene products in the receptor to achieve therapeutic purposes. Grafts may function in the receptor through two mechanisms:
A. Producing an active enzyme that is metabolized in situ to remove the original storage substrate.
B. Release active enzymes, coenzymes or immunologically active factors into the blood and distribute them to other tissues throughout the body. The organs and organs that have been transplanted so far have been kidney, liver, adrenal gland, bone marrow, thymus, spleen, pancreas, etc., and some have achieved remarkable effects.
4 enzyme replacement therapy: provide the corresponding normal enzyme directly to patients with enzyme deficiency. With the development of enzymatic technology and cell engineering and genetic engineering technology, a sufficient amount of high-purity enzyme preparation has been provided. This enzyme preparation must have characteristics such as long half-life, low antigenicity, and good orientation. The method commonly used for this is:
A. The enzyme preparation is packaged by using a carrier such as a microcapsule, a liposome or a red blood cell shadow to reduce the immunogenicity and prolong the half life.
B. Application of receptor-mediated molecular recognition to improve directivity.
C. For some lysosomal storage diseases, because the sediment can be diffused into the blood and maintain dynamic balance, it can be treated by the "balance-removal" method.
(3) Gene therapy: Gene therapy refers to a new treatment method that directly uses genetic transfer technology to introduce genetic material into germ cells or somatic cells to treat genetic diseases and other diseases. Gene therapy for genetic diseases is expected to fundamentally correct phenotypic abnormalities in genetic diseases.
1 Basic strategy of gene therapy: In the past ten years, gene therapy research has been flourishing, and many new ideas and new ideas have been proposed. The main strategies are:
A. In situ correction and in situ replacement of the gene, the purpose of this strategy is to repair the mutated gene in situ without affecting the structure and function of other genes surrounding it. Among them, in situ correction of point mutations or small-scale mutations for genes is proposed to be fixed by specific methods. In situ replacement, it is necessary to remove a gene with a large range of variation and replace it with a normal gene. This strategy is the most ideal and most direct method to cure genetic variation. Currently, many mammalian intracellular site-directed integration (homologous recombination) has provided theoretical and experimental evidence for this strategy, but it has not been true yet. For human testing.
B. Gene augmentation or gene complementation, transferring the exogenous functional gene into the diseased cell or individual genome without modifying the defective gene itself, and expressing it to compensate for the loss of the diseased gene. This strategy is currently the most studied and the most mature method.
C. Introducing an antisense gene or other gene that targets an abnormal gene expression product into a cell, and suppressing it, or gene inhibition therapy or intercellular immunity.
2 Technical points of gene therapy The most researched and matured strategies in gene therapy are applied to clinical trials. The entire research process usually includes preclinical studies and clinical studies (Table 1).
A. Choice of disease: The first choice for gene therapy is single-gene deficient disease. The basic conditions of selection often include:
a. The genetic basis is relatively clear, and the target gene can be cloned in vitro.
b. Gene expression does not need to be finely regulated, and is often open, and the physiological level of the product is not high.
c. It has a certain incidence rate, which is harmful, and there are still other effective treatment measures.
China is one of the countries that carried out research on gene therapy earlier. Xue Jinglun of Fudan University, etc., based on these conditions, chose hemophilia as a research object, and has achieved good results and reached the world advanced level. Of course, these conditions are limited to the current level of research.
B. Selection of target cells: Target cells for gene therapy can be divided into two major categories: germ cells and somatic cells. This led to the classification of germ cell gene therapy and somatic gene therapy. If genetic repair or replacement of germ cells or early embryonic cells can be made, and genetic defects can be corrected, genetic diseases can be treated not only in the contemporary age, but also can transmit new genes to the next generation, and also reduce a harmful gene for the population. The ideal means of radical cure for genetic diseases. However, due to modern biotechnology, theoretical limitations, and germ cell genetic manipulation involving many factors such as ethics, morality, and law in human society, animal testing can only be performed for a considerable period of time. In 1985, the US government had stipulated that human trials of gene therapy should be restricted to somatic cells. The target cells have been used: hematopoietic stem cells, hepatocytes, fibroblasts, endothelial cells, lymphocytes, and the like.
C. Vector transfer and transfer methods: constructing appropriate transfer and expression vectors and selecting efficient gene transfer methods are the key to gene therapy. Commonly used vectors are: retroviral vector, plasmid vector and adenoviral vector, adeno-associated virus The vector additionally has a liposome carrier. There are four main types of commonly used gene transfer methods:
a. Chemical method: mainly calcium phosphate precipitation method.
b. Physical method: commonly used conductance and microinjection.
c. Membrane fusion method: better by liposome encapsulation method.
d. Viral method: mainly refers to retrovirus and adenovirus-mediated gene transfer.
3 Prospects of gene therapy: The concept of gene therapy has been published for decades. It has only been nearly 10 years. With the development of modern molecular biology techniques (especially DNA recombination technology), this concept has gained a powerful theory. Fundamental and technical methods are supported and implemented. In 1990, two patients with severe immunodeficiency caused by adenosine deaminase (ADA) deficiency were successfully treated with gene therapy, marking a new phase in gene therapy research. Since then, biomedical scientists from all over the world have carried out research on gene therapy in an all-round way with the support of various government departments and various social forces. From the original development of a single genetic disease to a variety of diseases such as tumors and infectious diseases, new concepts and new approaches such as gene regulation therapy and gene suppression therapy have been proposed. By the first half of 1994, more than 100 clinical trial programs had been approved, and some had achieved good results. Of course, the history of gene therapy development is not long. It needs a lot of research and exploration to be widely used in clinical practice, especially the following aspects:
A. A deeper understanding of the molecular basis of more genetic diseases and the regulation mechanisms of gene expression, which is the basis of gene therapy.
B. Construct vectors that are expressed and transferred more efficiently and safely.
C. Establishment of a simpler and more efficient gene transfer method.
D. Perfection of technologies such as fixed point integration and in situ repair systems.
E. More close to the actual animal model (especially the transgenic animal model), which is the only way for preclinical testing of gene therapy.
F. The ethics of somatic cell gene therapy, germ cell gene therapy, and related scientific and technological management legislation.
G. It is also necessary to fully consider the possible harm of gene therapy, such as the serious consequences caused by the insertion mutation, the recovery of the defective viral vector after reconstitution, and other potential hazards of foreign gene transfer into the body. In short, we believe that gene therapy, as the only one that starts from the genetic defect itself, is expected to completely cure new treatments for genetic diseases. It has a very attractive future, but it still needs to be deepened from basic theory, technical methods and ethics. Extensive research and exploration can adapt to the modern medical model and be accepted by people, and truly become an effective means for human disease prevention and treatment.
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